Developmental sex-specific transcriptomic patterns can be altered by transgenic expression of Uty

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Rock, Lillian Folts, Hannah C. Zierden, Ruth Marx-Rattner, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2928137/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Nov, 2023 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract The genetic material encoded on X and Y chromosomes provides the foundation by which biological sex differences are established. Epigenetic regulators expressed on these sex chromosomes, including Kdm6a ( Utx ), Kdm5c , and Ddx3x have far-reaching impacts on transcriptional control of phenotypic sex differences. Although the functionality of UTY (Kdm6c , the Y-linked homologue of UTX), has been supported by more recent studies, its role in developmental sex differences is not understood. Here we test the hypothesis that UTY is an important transcriptional regulator during development that could contribute to sex-specific phenotypes and disease risks across the lifespan. We generated a random insertion Uty transgenic mouse (Uty-Tg) to overexpress Uty . By comparing transcriptomic profiles in developmental tissues, placenta and hypothalamus, we assessed potential UTY functional activity, comparing Uty -expressing female mice (XX + Uty) with wild-type male (XY) and female (XX) mice. To determine if Uty expression altered physiological or behavioral outcomes, adult mice were phenotypically examined. Uty expression masculinized female gene expression patterns in both the placenta and hypothalamus. Gene ontology (GO) and gene set enrichment analysis (GSEA) consistently identified pathways focused on transcriptional regulation, immune response, and lipid homeostasis as biological processes associated with UTY. Interestingly, adult females expressing Uty gained less weight and had a greater glucose tolerance compared to wild-type male and female mice when provided a high-fat diet. Utilizing a Uty -overexpressing transgenic mouse, our results provide novel evidence as to a functional transcriptional role for UTY in developing tissues, and a foundation to build on its prospective capacity to influence sex-specific developmental and health outcomes. Biological sciences/Developmental biology Biological sciences/Neuroscience Uty Kdm6c Sex Differences Transcriptional Regulation Placenta Hypothalamus Development Metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Sex differences in physiology, morphology, and behavior exist across the lifespan and contribute to significant differences in disease risk and resilience 1 . Established in early development, these differences including primary sex determination (i.e., gonadal) arise as the product of sex hormones and sex chromosomes where, in most cases, females are XX and males are XY 2 . Genes expressed on the X and Y chromosomes undoubtedly play a significant role in the timing and progression of sex-specific development, especially for the brain 2 – 6 . For example, studies using a unique rodent model known as the four core-genotypes (4CG), in which sex chromosome complement (XX vs XY) is distinct from gonadal sex due to autosomal displacement of the Sry gene from the Y chromosome, have highlighted masculinized neurological phenotypes in animals that are phenotypically female (female gonads), but genotypically male (XY) 7 , 8 . How specific genes, especially on the Y chromosome, impart sex-specific differences in tissue development and function is still largely unknown. Many X- and Y-linked genes are important in broadly controlling transcriptional responses critical to normal developmental processes 9 , 10 . However, the sex specificity to such regulatory mechanisms is less understood. Ubiquitously transcribed X chromosome tetratricopeptide repeat protein, or UTX, is a widely expressed X-linked demethylase 11 – 14 . UTX demethylates di- and tri-methylated histone 3 lysine 27 (H3K27me2/3), promoting a euchromatin state or active state of transcription (Fig. 1 A) 15 , 16 . Few studies have probed the biological function of UTY, the Y-linked homologue of UTX. Unlike UTX, current in vitro and in vivo evidence suggests that UTY lacks demethylase activity due to missing sequence within its catalytic domain 13 , 16 , 17 . However, recent studies provide evidence that UTX and UTY do share some functional redundancy, refuting the assumption that UTY is a nonfunctional remnant of UTX, and play an important role in differentiation, development, and pathophysiology, including cancer and heart disease 16 , 18 – 28 . For example, male Utx knock-out (KO) mouse embryos express normal levels of Uty and survive until birth, similar to heterozygous Utx females, while homozygous Utx -KO is embryonically lethal to females, suggesting UTY is functionally compensating for some aspects required for development in the absence of UTX in males. Further interrogation of UTY function in vitro previously demonstrated that redundancy in function did not appear to be specific to H3K27 demethylation, but rather was associated with other proteins and protein complexes that broadly regulate transcription 16 , 18 . Interestingly, discordance also exists in brain Utx and Uty expression patterns, where male mice showed much higher expression of Uty than Utx in the hypothalamus, suggesting a potential novel role for UTY in neuroendocrine regulation 29 . Still, there remains a paucity of data as to our understanding of functional roles of UTY, especially during fetal development in tissues where sex-specific regulation would be important, such as the placenta and hypothalamus. Recent studies from our lab and others have demonstrated that sex differences in transcriptional regulation in the placenta can be partially attributed to differential expression of the X-linked gene O-linked N-acetylglucosamine transferase ( Ogt ) between males and females 30 – 33 . Furthermore, sex differences in OGT dependent regulation of the H3K27 methyl transferase enhancer of zeste homologue 2 (EZH2) contributes to female resilience to the programmatic effects of prenatal stress (Fig. 1 A) 31 , 32 , 34 . However, the contribution of Y-linked genes to dissimilarities in placental transcriptional regulation has yet to be explored and may provide novel insight as to the gestational mechanisms conferring male-specific disease risk. Here we test the hypothesis that UTY is involved in regulation of male-specific gene expression using the placenta and hypothalamus as important developmental tissues with known sex differences in form and function. To examine a possible functional action of UTY, we developed a transgenic mouse with a random insertion of the full Uty gene (Uty-Tg; Fig. 1 B). An unbiased transcriptomic approach was used as a proxy for UTY function by comparing female mice with Uty expression (XX + Uty) to wild-type male (XY) and female (XX) mice, utilizing transcriptomics tools for a conceptual methodological examination for broad gene expression changes, e.g., GSEA and DAVID, as evidence for UTY function 35 – 39 . To probe changes in UTY demethylase activity and function in our transgenic mice, we utilized our previously published H3K27me3 ChIP-Seq dataset to assess overlap in UTY gene regulation with male-specific H3K27me3 binding to examine the potential role of UTY in transcriptional repression 34 . Finally, we examined adult phenotypic outcomes relevant to alterations in hypothalamic function, including the HPA stress axis and feeding and body weight gain in response to a calorically dense dietary challenge. Methods Animals All experiments were approved by the University of Maryland School of Medicine and the University of Pennsylvania Institutional Animal Care and Use Committees and performed in accordance with the National Institutes of Health Animal Care and Use Guidelines. Animal experiments are reported in accordance with ARRIVE guidelines. The recombinant Uty DNA construct (180Kb) was generated by Gen-H, Genetic Recombineering Heidelberg, using a BAC clone backbone, floxed STOP cassette in the 5’UTR, and the entire Uty gene, including the 1.35 Kb of the endogenous promoter within the 20 Kb upstream of the transcriptional start site and all 27 exons ( Supplemental File 1 ). Transformation, selection, and injection of embryonic stem cells carrying this linear DNA construct into the inner cell mass of C57BL/B6 mouse blastocysts was performed at the School of Veterinary Medicine, University of Pennsylvania to establish the Uty transgenic strain (Uty-Tg). All Uty-Tg mice were derived from successful random integration into a single clone, which we acknowledge is a limitation of this study. However, the absence of profound changes to the transcriptome under control conditions as shown and discussed below, suggest that our results were not significantly impacted by the initial BAC integration (Fig. 1 C and 1 D). While a conditional Uty-overexpressing mouse was our original intended approach, in our examination of Uty tissue overexpression, we unfortunately confirmed a lack of tissue specificity and thus we are assessing outcomes as a global Uty -overexpressing mouse ( Supplemental File 1 and 2 ). Relative expression of Uty mRNA was validated in the E18.5 XX + Uty fetal placenta and brain and demonstrated that Uty-Tg animals express Uty at levels that were equal to or greater than wild-type male endogenous expression levels (i.e., Uty -overexpressing; Supplemental File 2 ). Mice were housed under a 12h light/day photoperiod with lights on at 0700 EST and ad libitum access to water and grain-based chow diet (Purina Rodent Chow 5001, St. Louis, MO). Dams (50:50 C57BL/B6:129-CYP19-Cre, PCre) were paired with (C57BL/B6-Uty-Tg) sires for the initial breeding to generate offspring that were on a B6:129 background. For fetal collections dams and sires were paired overnight, starting at 1700 and separated at 0700 EST and checked for a copulation plug 40 . Noon on the day that the plug was observed was considered embryonic day 0.5 (E0.5). Fetal collections took place at E18.5 and tissues were collected for genotyping and RNA-sequencing ( Supplemental File 2 ). Fetal tissues used for sequencing were all first generation on a B6:129 background and did not express PCre ( Supplemental File 2 ). Several primer combinations were tested identifying the primers P587-check1 (‘5-CACTGGTGATGACGCAAGTC-3’) and GBPR281 (‘5-CACCACTGCTCCCATTCATC-3’) as the optimal combination for consistent Uty-Tg genotyping ( Supplemental File 3 ). Embryonic sex determination was achieved by genotyping using primers specific for Jarid1 (‘5-TGAAGCTTTTGGCTTGAG-3’ and ‘5-CCGCTGCCAAATTCTTTGG-3’) as previously described 41 . For offspring phenotypic assessments, dams were allowed to litter, and pups remained with their mothers undisturbed until weaning at postnatal day 28 (PN28). Tissue Collection For fetal collections, pregnant dams were euthanized at E18.5 and fetal tails, heads, and placentas were collected. DNA was isolated from fetal tails for genotyping and sexing. One XY male, one XX female, and one XX + Uty female were used for each analysis to eliminate potential confounding litter effects. Fetal heads and hemisected (i.e., halved) placentas were placed in cryotubes, and frozen in liquid nitrogen. Tissues were stored at -80℃ until RNA isolation. RNA Sequencing and Analysis E18.5 fetal heads were cryosectioned at 300 µm, -20℃ on a Cryostat. Using a 1 mm tissue punch, two 1mm x 300 µm thick punches were collected that corresponded to Figs. 63–68 in the P0 section of the Atlas of the Developing Mouse Brain to obtain a punch enriched for the paraventricular nucleus (PVN) of the hypothalamus, referred to as the hypothalamus throughout the manuscript 42 . Micropunches were immediately dispensed into 500 µl of Trizol and stored at -80℃ until RNA isolation. Placentas were homogenized in 500 µl of Trizol in preparation for RNA extraction and purification. For both the hypothalamus and placenta, RNA was extracted from tissue/Trizol homogenates via chloroform liquid-liquid extraction and alcohol precipitation. Purification of mRNA was performed using Qiagen RNeasy Mini kits and Illumina cDNA libraries of E18.5 hypothalamus and placental mRNA were prepared from 25 ng and 300 ng total RNA, respectively, using the Illumina Stranded mRNA Prep kits (20040532, Illumina) and indexes (20040553, Illumina) according to the manufacturer's protocol. Library fragment size was quantified using Agilent High Sensitivity D1000 ScreenTape Assays (5067–5584, Agilent). Library concentrations were determined using the Qubit dsDNA High Sensitivity Assay (Q32851, ThermoFisher). Samples were multiplexed and sequenced on an Illumina NextSeq550 instrument using high output 1x75bp chemistry (20024906, Illumina). FASTQ files generated from Illumina were concatenated and used as an input to kallisto, a pseudoalignment program, for alignment to the Mus musculus reference transcriptome (version 38) 43 . The remaining analysis took place in the R statistical environment (Version 4.1.1; R Core Team 2021). Gene isoforms were assigned to gene symbols using the Bioconductor package tximport and genes were filtered to counts per million > 1 in at least four samples for the hypothalamus and > 1 in at least five for placental analysis 44 . One wild-type female sample was removed from the hypothalamic analysis due to an issue with library preparation. The filtered gene lists were then normalized using trimmed mean of M-values in edgeR, variance weights were calculated using voom, and differential expression analysis of linear fit models was performed using limma, Benjamini-Hochburg false discovery rate (FDR) 1 45–47 . Although we did not observe significant differences in individual genes based on conservative corrections for multiple comparisons, adjusted p ≤ 0.05, differential expression of genes with uncorrected p ≤ 0.05 were assessed as a hypothesis generating approach for future studies. The remaining analyses were performed as gene set or cluster-based analysis as we hypothesized that expression of Uty would likely have a significant overall impact on expression of numerous genes and biological processes, as is the function of a GSEA. method. First, heatmaps were generated with all detectable transcripts using unbiased hierarchical clustering of genes based on z-scores for differential gene expression between XX + Uty and XX females (contrast matrix XX + Uty female - XX female) for each tissue. Modules were determined from a row cluster height of 0.3 that resulted in 12 different modules in both the placenta and hypothalamus. The Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resource was used to analyze gene sets within these modules in order to identify and understand potential biological relevance from broad changes in gene expression associated with Uty expression using GO subsets biological processes (BP), molecular function (MF), and cellular component (CC) and significance threshold set at FDR 1 38,39 . Gene set enrichment analysis (GSEAv4.3.2, Broad Institute, Cambridge, MA) of normalized counts were used to identify patterns of gene expression and determine greater-than-chance enrichment of biological pathways in a threshold-free manner (i.e. without consideration for differential gene expression), as previously reported by our work and that of other labs 35 , 37 , 48 . Briefly, collections of GO subcategory biological processes (GO:BP) annotated gene sets were obtained from the Molecular Signature Database (MSigDBv7.4.1, Broad Institute, Cambridge, MA) available for use with GSEA software. Gene set permutations were computed in GSEA to determine FDR, nominal p value, and normalized enrichment score (NES) of each gene set with a significance threshold set at FDR 1. Phenotypes To communicate the practical significance (i.e., magnitude of effect), effect sizes were calculated for all phenotypic measures. ANOVA effect size was determined by calculating Eta squared (η 2 ), effects of which are defined as small at 0.01, medium at 0.06, and large at 0.14. Effect sizes for post-hoc Tukey’s or Sidak’s corrections for multiple comparisons were calculated by Cohen’s d, effects of which are defined as small at 0.2, medium at 0.5, and large at 0.81 49 . Animal Weights For all studies in adult animals, first generation offspring on a B6:129 mixed background received a unique identification ear tag at PN28 and were weighed weekly from PN28 - PN70. At PN70, animals were placed on a high-fat diet (HFD; Research Diets Inc, D12492), calories provided by protein 29% and fat 14%, or remained on the grain-based control chow diet (Purina Rodent Chow 5001, St. Louis, MO), calories provided by protein 20% and fat 60%, to assess phenotypic outcomes following a 5-week dietary challenge. Two-way repeated measures and one-way ANOVAs, with Sidak’s or Tukey’s multiple comparisons tests, respectively, were conducted using Prism (Graphpad), α = 0.05. Corticosterone Response to Restraint Stress PN56 XX, XY, and XX + Uty offspring underwent restraint stress by being placed in a 50 mL conical tube for 15 min. A single < 1mm distal tail snip was made at time 0 to collect 10 µL of tail blood at 0, 15, 30, and 120 minutes, which was placed in EDTA-treated tubes. Quantification of plasma corticosterone levels was determined by radioimmunoassay (207120, MP Biomedicals). Two-way repeated measures ANOVA with Sidak’s multiple comparisons post-hoc test was conducted using Prism (Graphpad), α = 0.05. Food Consumption Food consumption was assessed at PN84 for a 24 hr period by weighing food at hour 0 and again at hour 24. Animals were pair-housed in the same genotype pairs for this assessment and data was analyzed as weight of food consumed per animal, assuming each animal ate the same amount of food, and normalized to individual body weight. A one-way ANOVA with Tukey’s multiple comparisons post-hoc test was conducted using Prism (Graphpad), α = 0.05. Glucose Tolerance Test A glucose tolerance test was administered at PN91 to XX, XY, and XX + Uty offspring. Animals were fasted for six hours before receiving an intraperitoneal injection of 0.3g/ml glucose in saline. A Contour Next Blood Glucose Monitoring System (Bayer Co, Germany) was used to measure blood glucose levels. A single < 1 mm distal tail snip was made to collect tail blood at 0, 30, 60, and 120 min from the time of injection. Two-way repeated measures ANOVA with Sidak’s multiple comparisons post-hoc test was conducted using Prism (Graphpad), α = 0.05. Data Availability The RNA-sequencing data generated in this study has been deposited in the NCBI SRA database under accession code: PRJNA883432 . Results Placenta and Hypothalamic Gene Expression Cluster dendrograms and principal components analysis (PCA) showed little separation between XX + Uty and XX females in the placenta (Fig. 1 C and 1 D) or hypothalamus (Fig. 1 E and 1 F). Differential gene expression analysis (contrast matrix XX + Uty female - XX female; FDR 1) revealed no significant changes in individual gene expression, with the exception of Uty , between the genotypes in the placenta or hypothalamus. Additionally, expression of Utx was not altered by overexpression of Uty in XX + Uty animals ( Supplemental File 2 ). Collectively, these findings support an interpretation that changes in overall gene expression in XX + Uty animals compared to XX did not the result from our random insertion overexpressing transgenic approach. However, as a random insertion method does not allow for confirmation of where or how many insertions in the genome have occurred, we acknowledge this potential methodological confound. Differential gene expression analysis was used to generate volcano plots using unadjusted p-values and logFC to identify potential genes of interest associated with Uty-Tg expression. Uty was the only gene that reached statistical significance by adjusted p-value in both the placenta (p = 0.001) and hypothalamus (p < 0.0001), with increased expression observed in XX + Uty tissues compared to XX females (Fig. 2 ). In the placenta and hypothalamus, we identified 618 and 777 genes, respectively, that had unadjusted p ≤ 0.05. A handful of up- and downregulated genes are highlighted, with one specific gene, Cep350 , downregulated in both the placenta and hypothalamus (Fig. 2 A and 2 B). Hierarchical clustering was performed on all detectable transcripts without p-value or logFC restrictions (p = 1, logFC = 0) to look at broad differences in gene expression profiles between XX + Uty, XX, and XY animals. In the placenta, there were a total of 14,230 detectable transcripts that were used for hierarchical clustering and grouped into twelve distinct modules. We identified modules with differential gene expression patterns that were directionally similar (i.e., similarly upregulated or downregulated based on z-score) for XX + Uty compared to XX females, similar for XX + Uty females and XY males compared to XX females, as well as modules with differential expression that was unique to XX + Uty (e.g., upregulated in XX + Uty but downregulated in XX and XY), or expression profiles that did not show a change in directionality of regulation (Fig. 3 A). Modules 4, 5, and 12 are referred to as masculinized because the direction of change in XX + Uty females relative to XX females is the same as the direction of change in XY males relative to XX females. The number of genes in these modules corresponded to 21.3% differential expression XX + Uty and XX, 16.4% differential expression XX + Uty and XY, 49.4% differential expression XX + Uty, and 12.9% without overlapping expression patterns between genotypes. Percentages were based on the total number of detectable transcripts (Fig. 3 B). Enriched biological processes were identified using DAVID functional annotation tools. For analysis, gene lists from modules that showed similarities in directionality of change were grouped together, with modules 2, 7, and 11 as a group and modules 1, 8, and 9 as another group, and input into DAVID separately to assess downregulated (blue) and upregulated (red) biological processes, FDR 1. Processes enriched in the XX + Uty modules included protein modification, through kinase, phosphatase, transferase, and peptidase enzymes, ribosomal structure and translation, and extracellular matrix assembly. The same approach was used to identify biological processes in the masculinized XX + Uty female gene sets, which showed enrichment for biological processes including kinase and transferase activity, vesicle mediated transport, cholesterol homeostasis and lipid transport, fibrinogen complex, and mRNA processing (Fig. 3 C). Finally, GSEA analysis of normalized counts was performed, with FDR 0 ~ XX + Uty phenotype, and NES < 0 ~ XX female phenotype ( Supplemental Data 1 ). Dot plots represent the top 20 significantly enriched biological processes that included inflammatory and microbial response pathways, extracellular matrix disassembly, and regulation of peptidase and hydrolase activity ascribed solely to the XX + Uty phenotype (Fig. 3 D). In the hypothalamus, the total number of detectable transcripts was 14,553, which were used for hierarchical clustering and grouped into twelve distinct modules. Similar to the placenta, we identified modules with differential gene expression patterns that were directionally similar for XX + Uty compared to XX females, similar for XX + Uty females and XY males compared to XX females, as well as modules with differential expression that was unique to XX + Uty (e.g., upregulated in XX + Uty but downregulated in XX and XY), or expression profiles that did not show a change in directionality of regulation (Fig. 3 E). Modules 9 and 2 are referred to as masculinized because the direction of change in XX + Uty females relative to XX females is the same as the direction of change in XY males relative to XX females. The number of genes in these modules correspond to 20.2% differential expression XX + Uty and XX, 24.8% differential expression XX + Uty and XY, 36.5% differential expression XX + Uty, and 18.5% without overlapping expression patterns between genotypes. Percentages are based on the total number of detectable transcripts (Fig. 3 F). Enriched biological processes were identified using DAVID as described for the placenta analysis above. Processes enriched in the XX + Uty modules included ion channel activity, microtubule-based movement, cell projection organization, mRNA processing, transferase activity, and regulation of transcription. The same approach was used to identify biological processes in masculinized XX + Uty modules. Biological processes enriched in these modules included synapse and cell junction, transferase activity and ATP-binding, calcium transport, cell cycle and division, and regulation of interferon alpha and beta (Fig. 3 G). GSEA was performed as described for the placenta analysis above. In the hypothalamus, biological processes enriched in the XX + Uty phenotype included response to wounding, chromosome segregation, and blood vessel and muscle morphogenesis. However, the XX female phenotype showed enrichment for synaptic signaling, hormone regulation, and feeding behavior (Fig. 3 H). Masculinized Gene Expression Patterns and Transcriptional Regulation in the XX + Uty Female Placenta and Hypothalamus Focusing on the genes that showed male-like differential expression patterns in XX + Uty tissues, or masculinized differential expression patterns, we probed for commonalities between tissues and utilized previously published data sets to assess a role for UTY in sex-specific gene expression and transcriptional regulation. While most of the masculinized differentially expressed genes were not shared between the placenta and hypothalamus, confirming a likely tissue-specific role for UTY, 260 genes did show similar differential expression patterns with 108 upregulated and 152 downregulated (Fig. 4 A). Due to the limited number of genes identified, we were not able to assess enriched biological processes in an upregulated or downregulated specific manner. Instead, DAVID was used to identify enriched biological processes in this list of 260 genes. Enriched processes included ATP-binding and transferase activity, protein phosphorylation, kinase activity, tyrosine activity, and fatty acid oxidation (Fig. 4 B). Using previously published datasets reporting transcriptomic sex differences in the placenta and hypothalamus, we compared our masculinized XX + Uty genes to determine if Uty played a broader role in masculinizing gene expression 34 , 50 . More specifically, we compiled a list of 229 placental genes and 182 hypothalamic genes that showed significant differences, p ≤ 0.05, in expression between XX females and XY males from these published datasets. In the placenta, we observed a 14% (32 genes) overlap in the placenta and a 21.4% (39 genes) overlap in the hypothalamus between previously reported sex differences in gene expression and masculinized XX + Uty genes (Figs. 4 C and 4 D). Of interest and developmentally important, we identified estrogen receptor alpha ( Esr1 ) and several estrogen responsive genes, oxytocin ( Oxt ), insulin-like growth factor 1 ( Igf1 ), and protein kinase C delta ( Prkcd ), in both our masculinized XX + Uty gene list and reported sex differences in the hypothalamus, suggesting a potential interaction between UTY and estrogen receptor transcriptional regulation 51 – 55 . The ability of UTY to function as a demethylase, similar to its X-linked homologue, UTX, has yet to be determined. We integrated our previously published H3K27me3 chromatin immunoprecipitation sequencing (ChIP-Seq) dataset from the placenta of mice on a B6:129 background with our masculinized XX + Uty female placental genes 34 . To test the hypothesis that the presence of UTX and UTY in male (XY) cells would contribute to increased H3K27me3 demethylation and therefore fewer H3K27me3 marks at transcriptional start sites, we first filtered the ChIP-Seq data accordingly. This process involved removing duplicate transcriptional start sites (TSS) from the ChIP-Seq dataset, identifying genes with reduced TSS counts in males (XY) compared to females (XX), and then comparing the list of genes to our masculinized XX + Uty gene list that included both upregulated and downregulated genes (Fig. 4 E). This process revealed 1155 genes shared between the filtered ChIP-Seq dataset and the masculinized XX + Uty gene list, resulting in 9.5% of all genes we had previously identified with H3K27me3 TSS counts where XY < XX and 49.5% of the masculinized XX + Uty gene list (Fig. 4 F). The biological processes enriched in the 1155 genes include transferase and kinase activity, nucleotide and ATP-binding, and lipid transport (Fig. 4 G). Uty Confers Vulnerability to Metabolic Dysregulation in HFD Females A significant effect of genotype was observed for baseline body weight measurements (F (2,29) = 63.4, p = 0.0001; η 2 = 0.81; Fig. 5 A), with males consistently heavier than XX + Uty (PN28, p = 0.02; PN35 – PN70, p ≤ 0.0001; d = 1.98) and XX females (PN35, p = 0.0002; PN42–70, p ≤ 0.0001; d = 1.81). No significant effect of Uty-Tg was observed for baseline female body weight measurements (Fig. 5 A). No main effect of genotype was observed for stress response measured by restraint stress and HPA activity (Fig. 5 B), however post-hoc analysis did reveal that XY males had significantly lower levels of corticosterone at 120 minutes compared to XX and XX + Uty females (p = 0.007; d = 0.65; p = 0.04; d = 0.56). When animals were challenged with a HFD, a significant effect of genotype was observed (F (2,29 = 85.08, p = 0.0001; η 2 = 0.85; Fig. 5 C), where males were heavier than XX + Uty (Wk1–5, p ≤ 0.0001; d = 3.70; Fig. 5 C) and XX females (Wk1–5, p ≤ 0.0001; d = 2.86; Fig. 5 C). Body weight assessments at the end of the 5 week dietary challenge revealed a significant effect of genotype (F(2,29) = 79.50, p = 0.0001; η 2 = 0.85; Fig. 5 D), with XX females weighing less than XY males (p ≤ 0.0001; d = 4.52; Fig. 5 D) and XX + Uty females weighing less than XY males and XX females (p ≤ 0.0001; d = 5.50 and p = 0.03; d = 1.08; Fig. 5 D). A significant effect of genotype was observed for percent weight gained on HFD over the five-week period (F (2,29) = 10.45, p = 0.0004; η 2 = 0.42; Fig. 5 E), with XX + Uty females consistently having a lower percent weight gained than XY males (Wk2, p = 0.01; Wk3, p = 0.0002; Wk4, p = 0.003; Wk5, p = 0.0004; d = 1.50; Fig. 5 E). This significant effect of genotype persisted to the end of the five-week dietary challenge (F (2,29) = 12.65, p = 0.0001; η 2 = 0.47; Fig. 5 F), where XX + Uty females had a significantly lower percent weight change than XY males and XX females (p = 0.02; d = 2.54; p ≤ 0.0001; d = 1.14; Fig. 5 F). To assess if these changes in body weight corresponded to changes in glucose regulation and food consumption, we ran a glucose tolerance test (GTT) and measured the 24-hour food consumption. Genotype had a significant effect on glucose tolerance (F (2,31) = 4.407, p = 0.02; η 2 = 0.22; Fig. 5 G), with XX + Uty females having significantly lower glucose levels at 60 and 120 minutes compared to XY males (p = 0.0002; p ≤ 0.0001; d = 0.48; Fig. 5 G). Finally, a significant effect of genotype was observed for 24-hr food consumption (F(2,11) = 11.87, p = 0.0018; η 2 = 0.68) with XY males consuming significantly more food compared to XX (p = 0.02; d = 2.08) and XX + Uty females (p = 0.0014; d = 4.89; Fig. 5 H). Discussion Sex differences in fetal development and sensitivity to intrauterine perturbations are well documented, however, there remains a paucity of information on specific molecular and/or genetic mechanisms responsible for divergent male and female development and health outcomes. We previously showed that OGT, a protein encoded by an X-linked gene, contributes to female fetal protection from prenatal stress, in part through its regulation of the H3K27 methyltransferase, EZH2 31,32,34 . Increased OGT is associated with higher levels of H3K27me3 in the female placenta, in both mouse and human tissue 34 . However, less is understood as to the sex-specific roles of the X- and Y-linked H3K27 demethylases, UTX and UTY. Comparisons of UTX and UTY amino acid sequences demonstrate extensive sequence homology, reaching 88% homology, both within and outside of the catalytic domain, yet the function of UTY remains less clear 16 , 17 . Important to sex-differences in H3K27me3-mediated transcriptional regulation in the placenta, XY tissues naturally have reduced OGT in addition to the presence of UTY, potentially culminating in overall reduced levels of H3K27me3 and reduced transcriptional repressive control in tissues such as the placenta 34 . As such, we hypothesized that these differences may account for an increased vulnerability for males in utero, especially for neurodevelopmental risk 4 – 6 , 56 . We previously reported no sex difference for placental Utx expression suggesting that Utx , unlike Ogt , may not escape X inactivation in the placenta 31 , 34 . Therefore, we hypothesized that if UTY indeed had transcriptional influence, either via potential demethylation of H3K27me3 and/or other as of yet unidentified activity, it would serve as a molecular mechanism that could direct male-specific fetal development. Therefore, we generated a Uty -overexpressing transgenic mouse and utilized a transcriptomic approach to assess potential changes in placental and fetal brain development. Initial phenotypic assessment found no overt developmental changes or outcomes in the XX + Uty mice. As Uty is not a master regulator (i.e., not known to be at the top of a gene regulation hierarchy) and limited protein-protein interactions have been described for UTY, we did not anticipate that over-expression of Uty in a wild-type female mouse would produce profound phenotypic changes. We examined global health outcomes in the XX + Uty mice and confirmed that they showed no signs of developmental abnormalities or health consequences resulting from Uty expression. Significant differences in gene expression based on conservative corrections for multiple comparisons and distinct differences in clustering assessed by cluster dendrogram and principal components analysis were not observed in the placenta or hypothalamus. We interpret these findings as an indication that we have not caused significant, potentially detrimental changes to the genome by the random insertion approach. Furthermore, the lack of baseline changes in body weight and functionality of the stress-axis (HPA) in XX + Uty compared to XX animals indicates that growth, metabolism, and neuroendocrine regulation were relatively normal, under control housing and feeding conditions. Utilizing our transcriptomic results as a hypothesis generating dataset, we adjusted our statistical parameters to examine relationships and patterns based on unadjusted p-values and in all detectable transcripts from our list of differentially expressed genes. We first compared gene sets between tissues to identify those that were similarly regulated in both. In the placenta and developing hypothalamus of XX + Uty animals, we found 206 genes that were enriched for biological processes including autophosphorylation and kinase activity. Autophosphorylation of protein kinases is a common process by which the enzyme adds a phosphate group to itself, altering its catalytic activity. Examples of such tyrosine kinases include epidermal growth factor receptor, insulin receptors, and SRC kinases that are important for proliferation, differentiation, and metabolism 57 , 58 . In addition to the shared biological processes, we also identified the gene for centrosome-associated protein 350 ( Cep350 ) as a similarly downregulated gene in XX + Uty placenta and hypothalamus. CEP350 is required for the anchoring of microtubules and recruitment of some nuclear receptors, such as peroxisome proliferator-activated receptor alpha (PPARα), a major regulator of lipid metabolism. Centrosomal genes, including Cep350 , play a pivotal structural role in primary cilia that are antenna-like sensory organelles found in the placenta and hypothalamus to coordinate developmental processes and metabolic homeostasis 59 – 61 . Furthermore, defective cilia have been implicated in the pathogenesis of preeclampsia and metabolic disorders 60 , 61 . Dysregulation of Cep350 associated with Uty expression likely deserves further interrogation as a male-specific source of vulnerability for pregnancy complications and metabolic dysregulation. These overlapping gene sets in the placenta and hypothalamus of XX + Uty animals consistently suggest an association between Uty expression and regulation of genes important for metabolism. Unique to the placenta, almost 50% of the 14,230 detected transcripts in the placenta showed differential expression profiles that were completely distinct to XX + Uty animals. Gene ontology (GO) identified biological processes related to transcription, translation, and post-translational protein modifications, suggesting that UTY plays a regulatory role in gene expression and protein synthesis and modification. Not surprisingly, we saw a ~ 20% overlap in gene regulation between XX + Uty and XX females, but we focused on the potential masculinizing effects of UTY on gene regulation, (i.e., pattern similarities between XX + Uty and XY mice), as evidence of novel UTY function. We found that 16.4% of detectable transcripts were male-biased in their expression pattern in the placenta where biological processes vital to cellular signaling, metabolism, and inflammation were enriched, including protein ubiquitination 50 . Ubiquitination is a common post-translational protein modification that tags proteins for sorting, localization, trafficking, and degradation. Therefore, protein ubiquitination is essential to both intracellular and extracellular communication that can impact diverse cellular processes such as DNA transcription, cell cycle, ribosome biogenesis, and inflammation. For example, new evidence suggests that ubiquitinated proteins are directed toward extracellular secretion via membrane-bound vesicles known as extracellular vesicles (EVs). EVs serve as carriers for a variety of proteins, microRNAs, mRNAs, and lipids transporting these biological molecules to neighboring cells and mediating physiological processes involved in development and homeostasis 62 . While the physiological consequences of altered protein ubiquitination associated with Uty expression are impossible to discern without further studies, it may be one underlying mechanism that contributes to several other biological processes enriched in our masculinized XX + Uty female dataset, including vesicle transport, cholesterol homeostasis, and inflammation. Regulation of immune tolerance is essential for successful pregnancy and relies on coordination and communication between neighboring cells. The semi-allogeneic placenta derives half of its genetic material from the mother and half from the father which leads to the expression of foreign, paternal, proteins in the intrauterine environment 63 , 64 . Therefore, several mechanisms are in place to protect the placenta from being targeted by the maternal immune system 65 . We observed alterations in the regulation of pathways associated with inflammatory and immune responses in placentas with Uty expression that raises some questions regarding the role of UTY in chronic placental inflammation, a phenotype more often observed with male fetuses and associated with premature delivery 66 , 67 . Other studies have reported associations between experimentally reduced Uty expression and changes in immune response, although there are inconsistencies in the directionality of immune dysregulation 68 , 69 . Disruption of cholesterol homeostasis also deserves further interrogation as a pathway that is partially regulated by UTY in the placenta. The placenta acquires cholesterol from maternal circulation and synthesizes cholesterol that is subsequently transferred to the fetus or used by the placenta to make hormones. Cholesterol levels in healthy pregnancies increase over time. However, too much cholesterol is associated with increases in lipid peroxidation, reactive oxygen species, and inflammation, as well as pregnancy complications including preeclampsia and fetal growth restriction 70 . These similarly regulated pathways between XX + Uty and XY placentas provide novel evidence that UTY might play a role in male-specific vulnerability to inflammation and pregnancy complications in utero . Unique to the hypothalamus, 14,553 transcripts were detected. Compared to the placenta, there were fewer genes distinct to the XX + Uty females (36.5%), and more genes with similar expression profiles between XX + Uty and XY (24.8%). We also detected a greater number of genes (39) overlap between the masculinized XX + Uty gene list and previously published male-biased genes in the developing mouse hypothalamus, including estrogen receptor alpha ( Esr1 ), and several estrogen responsive genes, insulin-like growth factor 1 ( Igf1 ), oxytocin ( Oxt ), and protein kinase C delta ( Prkcd ) 34 , 51 – 55 . In the developing brain, testosterone aromatized to estradiol masculinizes the organization of neural circuits 71 – 76 . Thus, it is interesting to suggest that UTY may have a more ‘masculinizing’ effect on the hypothalamic transcriptome compared to the placenta. However, the biological pathways enriched in both the XX + Uty unique gene list and masculinized XX + Uty gene list were less specialized in the brain and more broadly related to the regulation of gene expression, protein phosphorylation, and RNA splicing and processing that may reflect cell type specific and/or timing of analysis outcomes. Our findings, along with recently reported evidence that UTY plays a role in male-specific neural stem cell differentiation, demonstrate that UTY may be a contributing molecular mechanism by which sex differences in brain organization arise 23 . The capacity for UTY to function as an H3K27me3 demethylase, like its X-linked homologue UTX, has been scrutinized for decades 15 , 16 , 18 , 77 . Utilizing previously published data from our lab, we integrated corresponding genes that were identified by H3K27me3 ChIP-Seq with our masculinized XX + Uty placenta dataset 34 . More specifically, we limited the placental ChIP-Seq data set so that we were only examining genes with transcriptional start sites that had counts lower in males compared to XX females, with the assumption that these genes were demethylated in a more male-specific direction. Interestingly, a small percentage of the genes from the masculinized XX + Uty group (9.5%) overlapped with the filtered ChIP-Seq gene list, however, that small percentage accounted for nearly 50% of the total masculinized XX + Uty genes. From these data we are not able to determine whether UTY may participate in demethylation of H3K27me3 in a direct or indirect manner, however, these findings suggest that a substantial portion of the masculinized XX + Uty genes have a reduction in this transcriptional repressive mark in the presence of UTY. In examination of adult phenotypes for effects of UTY, while adult XX + Uty females showed no significant differences from XX females under normal control conditions, they presented with surprising alterations in body weight and glucose tolerance following a 5-week calorically dense dietary challenge. While we hypothesized that a detectable phenotype would resemble wild-type males, we observed that XX + Uty females did not present in a masculinized manner. Surprisingly, XX + Uty females at the end of the HFD exposure weighed less and had a greater glucose clearance compared to either wild-type males or females. As this phenotype was unique to XX + Uty females, we interpret this finding as a potential role for UTY in metabolic regulation that may be related to developmental changes or current adult activity of UTY and may be distinct from XY and XX mice as a result of conflicting or compounding effects of UTY on an XX background. In examination of food consumption as an explanation for body weight differences, we found that XX + Uty females in fact consumed fewer calories than males on the HFD. However, no significant differences were detected between XX + Uty females and XX females suggesting again unique interactions of UTY on an XX background that are impacting metabolic processes. Future investigations into the mechanisms and hormonal regulation driving this Uty -associated phenotype could provide important insight about sex differences in metabolism and risk of metabolic disorders. Conclusions Epigenetic regulators expressed from X and Y chromosomes have significant potential to impart broad sex differences in transcriptional regulation, and to contribute to sex-specific responses to environmental perturbations. In such studies, the functional role of UTY has largely been ignored, however, the few studies that do exist suggest that it retains biological functions that may be independent from H3K27me3 demethylation 16 , 18 . The data reported herein suggest that UTY does indeed play a role in male-specific transcription. We specifically focused on the placenta and hypothalamus for this study due to the plethora of evidence showing that these tissues respond to changes in the intrauterine environment in a sex-specific manner. While our transcriptomic analysis was tissue-specific, our expression of Uty was not. Therefore, an important limitation of our study is regarding the global overexpression of Uty and the broad changes in any tissue that could produce secondary effects in the placenta or brain, and vice versa. Moving forward it would be important to modify the Uty construct to conditionally express the transgene. Furthermore, the random insertion of transgenes rather than a targeted insertion approach can disrupt normal gene sequences and yield unpredictable phenotypes and is a potential confounding factor in our study. We recognize the importance of utilizing site-specific overexpression and knockout models in future studies to probe if deletion of Uty results in loss of a proposed functions in XY males. More work is needed to further characterize the molecular functions and contributions to phenotypic outcomes by UTY. In conclusion, these studies provide novel insight as to the potential roles of UTY in male-specific gene expression that may underlie sex differences in risk and resilience across the lifespan and provide a foundation to build on in future studies. Declarations Funding Research in this manuscript was supported by the National Institute of Child Health and Human Development under award number R01HD097093 (T.L.B). The published content is the responsibility of the authors and does not necessarily represent the views of the National Institutes of Health. Authors’ Contributions Conceptualization (TB, BN), experimental design (TB, KR, LF, HZ), experimental execution (KR, LF, HZ, RMR, BN, NAL), drafted the manuscript (KR), edited the manuscript (TB, LF, HZ, RMR, BN, KR). Ethics Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that there are no competing interests. Availability of Data and Material The RNA-seq data generated in this study has been deposited in the NCBI SRA database under accession code PRJNA883432 . All other raw data are available upon request by contacting the corresponding author, Dr. Tracy Bale. Acknowledgements The authors would like to thank Gen-H, Genetic Recombineering Heidelberg, for producing and validating the Uty-Tg construct. 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Zierden","email":"","orcid":"","institution":"University of Maryland School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"C.","lastName":"Zierden","suffix":""},{"id":201673135,"identity":"2a2d8036-d3d4-4b3e-a116-1884a58bea32","order_by":3,"name":"Ruth Marx-Rattner","email":"","orcid":"","institution":"University of Maryland School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruth","middleName":"","lastName":"Marx-Rattner","suffix":""},{"id":201673136,"identity":"987c57b9-27ec-456e-9072-48876a9a0203","order_by":4,"name":"Nicolae Adrian Leu","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nicolae","middleName":"Adrian","lastName":"Leu","suffix":""},{"id":201673137,"identity":"ab94a5d5-31c4-4fcc-8837-173e316327de","order_by":5,"name":"Bridget M. Nugent","email":"","orcid":"","institution":"University of Maryland School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bridget","middleName":"M.","lastName":"Nugent","suffix":""},{"id":201673138,"identity":"f72a30e2-ea74-4d3b-bc42-d2e9b03aa040","order_by":6,"name":"Tracy L. Bale","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIiWNgGAWjYBAC+wNg6g8DHwMzhMkgAaEYG3BoYYNQx4AMtgSYFrBqQloOAxk8BkRqkT788DMPwzF5Nvaej595au7Y88/uMX/4g8FGdsMBHFr40oyleRj+GLbxnN0szXPsWeKMO2cMGyQY0oxxauFhMGMG2sLYJpG7jZmH7XCCgUSOYYMBw+FE3FrYvwG1HLZvk3/zjJnn32F7sJYEhv94tPCAbDmc2CbBw8bM23aYcQNIywGGA/i0FEvOMTiW3MaTZiw5t+9w4owbaYUzGwySjWfidtjGD28q/tj2sx9++OHNt8P2/DOSN3z8UWEn24dDCwgwwWIECWCKoADGH/jlR8EoGAWjYKQDAKrEWZG6BsZGAAAAAElFTkSuQmCC","orcid":"","institution":"University of Maryland School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tracy","middleName":"L.","lastName":"Bale","suffix":""}],"badges":[],"createdAt":"2023-05-12 14:44:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2928137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2928137/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-47977-x","type":"published","date":"2023-11-30T15:01:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37329356,"identity":"ea8dc75e-b933-4f1a-8dba-98bf791a6c33","added_by":"auto","created_at":"2023-05-22 14:55:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8686092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAs evidence of targeted specific function of Uty-Tg, global changes in gene expression were not observed with random insertion of Uty-Tg in XX females. A\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic of enzymes involved in methylation (glycosylated EZH2) and demethylation (UTX and potentially UTY) of histone 3 lysine 27. \u003cstrong\u003eB) \u003c/strong\u003eSimplified schematic of \u003cem\u003eUty\u003c/em\u003etransgenic construct. See Supplementary File 1 for more details. \u003cstrong\u003eA) \u003c/strong\u003eand \u003cstrong\u003eB)\u003c/strong\u003e created with BioRender.com. \u003cstrong\u003eC) \u003c/strong\u003eCluster dendrogram and \u003cstrong\u003eD) \u003c/strong\u003eprincipal component analysis (PCA) plots of gene expression data demonstrate the distribution of XY, XX, and XX +Uty placenta (n = 5). \u003cstrong\u003eE) \u003c/strong\u003eCluster dendrogram and \u003cstrong\u003eF)\u003c/strong\u003ePCA plots of gene expression data demonstrating the distribution of XY, XX, and XX +Uty hypothalamus (n = 4-5). In both the placenta and hypothalamus XX +Uty mice cluster with XX animals.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/3c54adbfd61f24ffec155ff0.png"},{"id":37330498,"identity":"77b0d304-9c50-4bf8-bef9-e1f11fc33538","added_by":"auto","created_at":"2023-05-22 15:03:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6608598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eUty\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression alters gene expression in a tissue-specific manner. \u003c/strong\u003eVolcano plots of differentially expressed genes showing upregulated (red) and downregulated (blue) genes in XX +Uty \u003cstrong\u003eA) \u003c/strong\u003eplacenta (n = 5) and \u003cstrong\u003eB) \u003c/strong\u003ehypothalamus (n = 4 – 5) relative to XX animals. The volcano plot was used to identify 3-4 significantly upregulated and downregulated genes, as shown and described in the table. The dotted gray lines indicate p = 0.05 and the dotted orange and blue lines denote log-fold change equal to 1 or -1, respectively.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/85b895e15c8f35f1901b6364.png"},{"id":37330499,"identity":"f2f880c2-b94d-42b4-9697-7c380b5c5e4c","added_by":"auto","created_at":"2023-05-22 15:03:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5531127,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMasculinized XX +Uty\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene expression patterns, determined by similarly regulated clusters between XX +Uty females and XY males, were identified in the placenta and hypothalamus. Placenta \u003c/strong\u003e(n = 5)\u003cstrong\u003e: A)\u003c/strong\u003e Heatmap depicting all detectable transcripts in the placenta (14,230). Unbiased hierarchical clustering was used to group genes into distinct clusters and identify gene sets that are similar in directionality of their expression profiles between XX +Uty and XX (black), XX +Uty and XY (green, masculinized), differential expression unique to XX +Uty (purple), or expression profiles that did not show a unique or overlapping pattern between the groups (white). \u003cstrong\u003eB) \u003c/strong\u003ePie chart showing the percentage of all detectable transcripts that are represented by these clusters in the placenta. \u003cstrong\u003eC) \u003c/strong\u003eBiological processes, identified by DAVID functional annotations, enriched in upregulated (red) and downregulated (blue) clusters assigned to differential expression in XX +Uty (purple) and masculinized XX +Uty\u003cem\u003e \u003c/em\u003e(green) groups. \u003cstrong\u003eD)\u003c/strong\u003e Dot plot depicting biological processes identified by gene set enrichment analysis (GSEA). Bubbles represent normalized enrichment scores (NES) of a particular gene set, color indicates increased (red) or decreased (blue), color intensity denotes magnitude of change, and bubble diameter represents gene set size. \u003cstrong\u003eHypothalamus \u003c/strong\u003e(n = 4-5)\u003cstrong\u003e: \u003c/strong\u003eThe same approach and figures as above are provided for all detectable transcripts in the hypothalamus. \u003cstrong\u003eE) \u003c/strong\u003eheatmap depicting all detectable transcripts in the hypothalamus (14,553), \u003cstrong\u003eF) \u003c/strong\u003epie chart showing the percentage of all detectable transcripts that are represented by the clusters described above in the hypothalamus, \u003cstrong\u003eG)\u003c/strong\u003e biological processes identified by DAVID functional annotations, and \u003cstrong\u003eH)\u003c/strong\u003edot plot depicting biological processes identified by GSEA.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/a3c80fc0317c734f39193b7f.png"},{"id":37330500,"identity":"d68af85f-d0d2-49ed-86f3-c91c7d945661","added_by":"auto","created_at":"2023-05-22 15:03:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7663088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMasculinized XX +Uty genes are shared between tissues, with established sex differences, and with genes that have male specific H3K27me3 profiles. A)\u003c/strong\u003e Venn diagram comparing masculinized XX +Uty genes that have increased (upregulated) or decreased (downregulated) expression in the placenta and hypothalamus. \u003cstrong\u003eB) \u003c/strong\u003eBiological processes that are enriched in the similarly upregulated (108) or downregulated (152) gene sets in the placenta and hypothalamus. Pie charts depicting overlap in masculinized XX +Uty and previously identified and published sex differences in the \u003cstrong\u003eC)\u003c/strong\u003e placenta (n = 5) and \u003cstrong\u003eD)\u003c/strong\u003e hypothalamus (n = 4-5) (Mao et al., 2010; Nugent et al., 2018). Gene names, descriptions, and chromosomes on which they are located are detailed in the inserted table, with genes that were similarly upregulated (red) or downregulated (blue) indicated by the colored text. \u003cstrong\u003eE)\u003c/strong\u003e Schematic describing how previously published H3K27me3 ChIP-Seq data from male placentas was used and compared to genes that were masculinized in the XX +Uty\u003cem\u003e \u003c/em\u003egene set (Nugent et al., 2018). \u003cstrong\u003eF) \u003c/strong\u003eParts of a whole bar plot depicting the proportion of genes with TSS counts XY \u0026lt; XX (gray, 12,205 genes) and masculinized XX +Uty genes (green, 2334) that overlap (gray and green patterned, 1155 genes). \u0026nbsp;Overlapping genes represent 9.5% of the genes identified in the male H3K27me3 ChIP-Seq and 49.5% of the XX +Uty masculinized dataset. \u003cstrong\u003eG) \u003c/strong\u003eBiological processes enriched in the 1155 overlapping genes, identified by DAVID functional annotations.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/e1374ab3588a738bbb995472.png"},{"id":37329357,"identity":"5c408544-d6e6-4241-9eba-e93b8b956d11","added_by":"auto","created_at":"2023-05-22 14:55:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3788873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXX +Uty animals gain less weight and have a higher glucose tolerance when challenged with a high fat diet compared to XX and XY mice. A) \u003c/strong\u003eBody weights were collected weekly from PN28-70. A significant effect of genotype was observed for baseline body weight measurements (F (2,29) = 63.4, p = 0.0001; η\u003csup\u003e2 \u003c/sup\u003e= 0.81), with XY being consistently heavier than XX +Uty\u003cem\u003e \u003c/em\u003e(PN28, p = 0.02; PN35 – PN70, p ≤ 0.0001; d = 1.98) and XX (PN35, p = 0.0002; PN42 – 70, p ≤ 0.0001; d = 1.81). There was no significant effect of Uty-Tg on XX +Uty bodyweight (n = 8-12). \u003cstrong\u003eB) \u003c/strong\u003eRegulation of corticosterone levels time course was assessed following a 15-min restraint stress with sampling at 0, 15, 30, and 120 min. No significant effect of Uty-Tg was observed on XX hypothalamic-pituitary-adrenal-axis (HPA) response (n = 7-13). Following baseline assessments, mice were challenged with a high fat diet (HFD) for 5 weeks. Body weights were collected weekly while on the diet. \u003cstrong\u003eC)\u003c/strong\u003e A significant effect of genotype was observed throughout the 5-wk dietary challenge (F (2,29 = 85.08, p = 0.0001; η\u003csup\u003e2 \u003c/sup\u003e= 0.85), where XY mice were heavier than XX +Uty (Wk1 – 5, p ≤ 0.0001; d = 3.70) and XX mice (Wk1 – 5, p ≤ 0.0001; d = 2.86). \u003cstrong\u003eD) \u003c/strong\u003eAt the end of the 5-wk HFD challenge, there was still a significant effect of genotype (F(2,29) = 79.50, p = 0.0001; η\u003csup\u003e2 \u003c/sup\u003e= 0.85, with XX mice weighing less than XY mice (p ≤ 0.0001; d = 4.52) and XX +Uty weighing less than XY and XX mice (p ≤ 0.0001; d = 5.50 and p = 0.03; d = 1.08). \u003cstrong\u003eE) \u003c/strong\u003eA significant effect of genotype was observed for percent weight gained on HFD over the five-wk period (F (2,29) = 10.45, p = 0.0004; η\u003csup\u003e2 \u003c/sup\u003e= 0.42), with XX +Uty consistently having a lower percent weight gain than XY mice (Wk2, p = 0.01; Wk3, p = 0.0002; Wk4, p = 0.003; Wk5, p = 0.0004; d = 1.50). \u003cstrong\u003eF)\u003c/strong\u003e This significant effect of genotype persisted to the end of the five-wk dietary challenge (F (2,29) = 12.65, p = 0.0001; η\u003csup\u003e2 \u003c/sup\u003e= 0.47), where XX +Uty mice had a significantly lower percent weight change than XY and XX mice (p = 0.02; d = 2.54; p ≤ 0.0001; d = 1.14).\u003cstrong\u003e G) \u003c/strong\u003eGlucose tolerance test (GTT) was performed at 0, 15-, 30-, 60-, and 120-min. Genotype had a significant effect on glucose tolerance (F (2,31) = 4.407, p = 0.02; η\u003csup\u003e2 \u003c/sup\u003e= 0.22), with XX +Uty mice having significantly lower glucose levels at 60 and 120 minutes compared to XY mice (p = 0.0002; p ≤ 0.0001; d = 0.48). \u003cstrong\u003eH)\u003c/strong\u003e Food consumption per cage (n = 4-5 cages with 2 mice/cage) was measured by weighing food at 0 and 24 hr, and showed a significant effect of genotype (F(2,11) = 11.87, p = 0.0018; η\u003csup\u003e2 \u003c/sup\u003e= 0.68) with XY males consuming significantly more food compared to XX and XX +Uty females (p = 0.02; d = 2.08; p = 0.0014; d = 4.89).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/f589e47faa819935c28e2c3f.png"},{"id":47864277,"identity":"6832a3a4-7c20-4d58-ad42-e2a0575a7c18","added_by":"auto","created_at":"2023-12-08 15:58:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1656984,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/63da37f9-4359-419d-90b2-8487031386b2.pdf"},{"id":37329362,"identity":"64aa89e5-f9a7-4715-9713-5c1f97d43602","added_by":"auto","created_at":"2023-05-22 14:55:44","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":138708,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/c3f3398c389065aa91d41c40.xlsx"},{"id":37329363,"identity":"a0260ec6-82e2-4773-b1d9-dd12bd23c3b3","added_by":"auto","created_at":"2023-05-22 14:55:44","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2524595,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/74ed50242a544f8376908b6d.pdf"},{"id":37329358,"identity":"c502336f-825d-4fb1-867a-709e4223cbac","added_by":"auto","created_at":"2023-05-22 14:55:44","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1537418,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/85a00240dfac555f28194c46.pdf"},{"id":37329360,"identity":"ceaebc98-62e6-403f-9ee8-4a308b5fa51e","added_by":"auto","created_at":"2023-05-22 14:55:44","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":416827,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2928137/v1/6616311750c034607369def5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Developmental sex-specific transcriptomic patterns can be altered by transgenic expression of Uty","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSex differences in physiology, morphology, and behavior exist across the lifespan and contribute to significant differences in disease risk and resilience \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Established in early development, these differences including primary sex determination (i.e., gonadal) arise as the product of sex hormones and sex chromosomes where, in most cases, females are XX and males are XY \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Genes expressed on the X and Y chromosomes undoubtedly play a significant role in the timing and progression of sex-specific development, especially for the brain \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. For example, studies using a unique rodent model known as the four core-genotypes (4CG), in which sex chromosome complement (XX vs XY) is distinct from gonadal sex due to autosomal displacement of the \u003cem\u003eSry\u003c/em\u003e gene from the Y chromosome, have highlighted masculinized neurological phenotypes in animals that are phenotypically female (female gonads), but genotypically male (XY) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. How specific genes, especially on the Y chromosome, impart sex-specific differences in tissue development and function is still largely unknown. Many X- and Y-linked genes are important in broadly controlling transcriptional responses critical to normal developmental processes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the sex specificity to such regulatory mechanisms is less understood.\u003c/p\u003e \u003cp\u003eUbiquitously transcribed X chromosome tetratricopeptide repeat protein, or UTX, is a widely expressed X-linked demethylase \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. UTX demethylates di- and tri-methylated histone 3 lysine 27 (H3K27me2/3), promoting a euchromatin state or active state of transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Few studies have probed the biological function of UTY, the Y-linked homologue of UTX. Unlike UTX, current \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evidence suggests that UTY lacks demethylase activity due to missing sequence within its catalytic domain \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, recent studies provide evidence that UTX and UTY do share some functional redundancy, refuting the assumption that UTY is a nonfunctional remnant of UTX, and play an important role in differentiation, development, and pathophysiology, including cancer and heart disease \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. For example, male \u003cem\u003eUtx\u003c/em\u003e knock-out (KO) mouse embryos express normal levels of \u003cem\u003eUty\u003c/em\u003e and survive until birth, similar to heterozygous \u003cem\u003eUtx\u003c/em\u003e females, while homozygous \u003cem\u003eUtx\u003c/em\u003e-KO is embryonically lethal to females, suggesting UTY is functionally compensating for some aspects required for development in the absence of UTX in males. Further interrogation of UTY function \u003cem\u003ein vitro\u003c/em\u003e previously demonstrated that redundancy in function did not appear to be specific to H3K27 demethylation, but rather was associated with other proteins and protein complexes that broadly regulate transcription \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Interestingly, discordance also exists in brain \u003cem\u003eUtx\u003c/em\u003e and \u003cem\u003eUty\u003c/em\u003e expression patterns, where male mice showed much higher expression of \u003cem\u003eUty\u003c/em\u003e than \u003cem\u003eUtx\u003c/em\u003e in the hypothalamus, suggesting a potential novel role for UTY in neuroendocrine regulation \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Still, there remains a paucity of data as to our understanding of functional roles of UTY, especially during fetal development in tissues where sex-specific regulation would be important, such as the placenta and hypothalamus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRecent studies from our lab and others have demonstrated that sex differences in transcriptional regulation in the placenta can be partially attributed to differential expression of the X-linked gene O-linked N-acetylglucosamine transferase (\u003cem\u003eOgt\u003c/em\u003e) between males and females \u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Furthermore, sex differences in OGT dependent regulation of the H3K27 methyl transferase enhancer of zeste homologue 2 (EZH2) contributes to female resilience to the programmatic effects of prenatal stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, the contribution of Y-linked genes to dissimilarities in placental transcriptional regulation has yet to be explored and may provide novel insight as to the gestational mechanisms conferring male-specific disease risk. Here we test the hypothesis that UTY is involved in regulation of male-specific gene expression using the placenta and hypothalamus as important developmental tissues with known sex differences in form and function. To examine a possible functional action of UTY, we developed a transgenic mouse with a random insertion of the full \u003cem\u003eUty\u003c/em\u003e gene (Uty-Tg; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). An unbiased transcriptomic approach was used as a proxy for UTY function by comparing female mice with \u003cem\u003eUty\u003c/em\u003e expression (XX\u0026thinsp;+\u0026thinsp;Uty) to wild-type male (XY) and female (XX) mice, utilizing transcriptomics tools for a conceptual methodological examination for broad gene expression changes, e.g., GSEA and DAVID, as evidence for UTY function\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To probe changes in UTY demethylase activity and function in our transgenic mice, we utilized our previously published H3K27me3 ChIP-Seq dataset to assess overlap in UTY gene regulation with male-specific H3K27me3 binding to examine the potential role of UTY in transcriptional repression \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Finally, we examined adult phenotypic outcomes relevant to alterations in hypothalamic function, including the HPA stress axis and feeding and body weight gain in response to a calorically dense dietary challenge.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experiments were approved by the University of Maryland School of Medicine and the University of Pennsylvania Institutional Animal Care and Use Committees and performed in accordance with the National Institutes of Health Animal Care and Use Guidelines. Animal experiments are reported in accordance with ARRIVE guidelines. The recombinant \u003cem\u003eUty\u003c/em\u003e DNA construct (180Kb) was generated by Gen-H, Genetic Recombineering Heidelberg, using a BAC clone backbone, floxed STOP cassette in the 5\u0026rsquo;UTR, and the entire \u003cem\u003eUty\u003c/em\u003e gene, including the 1.35 Kb of the endogenous promoter within the 20 Kb upstream of the transcriptional start site and all 27 exons (\u003cb\u003eSupplemental File 1\u003c/b\u003e). Transformation, selection, and injection of embryonic stem cells carrying this linear DNA construct into the inner cell mass of C57BL/B6 mouse blastocysts was performed at the School of Veterinary Medicine, University of Pennsylvania to establish the Uty transgenic strain (Uty-Tg). All Uty-Tg mice were derived from successful random integration into a single clone, which we acknowledge is a limitation of this study. However, the absence of profound changes to the transcriptome under control conditions as shown and discussed below, suggest that our results were not significantly impacted by the initial BAC integration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). While a conditional Uty-overexpressing mouse was our original intended approach, in our examination of \u003cem\u003eUty\u003c/em\u003e tissue overexpression, we unfortunately confirmed a lack of tissue specificity and thus we are assessing outcomes as a global \u003cem\u003eUty\u003c/em\u003e-overexpressing mouse (\u003cb\u003eSupplemental File 1 and 2\u003c/b\u003e). Relative expression of \u003cem\u003eUty\u003c/em\u003e mRNA was validated in the E18.5 XX\u0026thinsp;+\u0026thinsp;Uty fetal placenta and brain and demonstrated that Uty-Tg animals express \u003cem\u003eUty\u003c/em\u003e at levels that were equal to or greater than wild-type male endogenous expression levels (i.e., \u003cem\u003eUty\u003c/em\u003e-overexpressing; \u003cb\u003eSupplemental File 2\u003c/b\u003e). Mice were housed under a 12h light/day photoperiod with lights on at 0700 EST and ad libitum access to water and grain-based chow diet (Purina Rodent Chow 5001, St. Louis, MO). Dams (50:50 C57BL/B6:129-CYP19-Cre, PCre) were paired with (C57BL/B6-Uty-Tg) sires for the initial breeding to generate offspring that were on a B6:129 background. For fetal collections dams and sires were paired overnight, starting at 1700 and separated at 0700 EST and checked for a copulation plug \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Noon on the day that the plug was observed was considered embryonic day 0.5 (E0.5). Fetal collections took place at E18.5 and tissues were collected for genotyping and RNA-sequencing (\u003cb\u003eSupplemental File 2\u003c/b\u003e). Fetal tissues used for sequencing were all first generation on a B6:129 background and did not express PCre (\u003cb\u003eSupplemental File 2\u003c/b\u003e). Several primer combinations were tested identifying the primers P587-check1 (\u0026lsquo;5-CACTGGTGATGACGCAAGTC-3\u0026rsquo;) and GBPR281 (\u0026lsquo;5-CACCACTGCTCCCATTCATC-3\u0026rsquo;) as the optimal combination for consistent Uty-Tg genotyping (\u003cb\u003eSupplemental File 3\u003c/b\u003e). Embryonic sex determination was achieved by genotyping using primers specific for Jarid1 (\u0026lsquo;5-TGAAGCTTTTGGCTTGAG-3\u0026rsquo; and \u0026lsquo;5-CCGCTGCCAAATTCTTTGG-3\u0026rsquo;) as previously described \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. For offspring phenotypic assessments, dams were allowed to litter, and pups remained with their mothers undisturbed until weaning at postnatal day 28 (PN28).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTissue Collection\u003c/h2\u003e \u003cp\u003eFor fetal collections, pregnant dams were euthanized at E18.5 and fetal tails, heads, and placentas were collected. DNA was isolated from fetal tails for genotyping and sexing. One XY male, one XX female, and one XX\u0026thinsp;+\u0026thinsp;Uty female were used for each analysis to eliminate potential confounding litter effects. Fetal heads and hemisected (i.e., halved) placentas were placed in cryotubes, and frozen in liquid nitrogen. Tissues were stored at -80℃ until RNA isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing and Analysis\u003c/h2\u003e \u003cp\u003eE18.5 fetal heads were cryosectioned at 300 \u0026micro;m, -20℃ on a Cryostat. Using a 1 mm tissue punch, two 1mm x 300 \u0026micro;m thick punches were collected that corresponded to Figs.\u0026nbsp;63\u0026ndash;68 in the P0 section of the Atlas of the Developing Mouse Brain to obtain a punch enriched for the paraventricular nucleus (PVN) of the hypothalamus, referred to as the hypothalamus throughout the manuscript\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Micropunches were immediately dispensed into 500 \u0026micro;l of Trizol and stored at -80℃ until RNA isolation. Placentas were homogenized in 500 \u0026micro;l of Trizol in preparation for RNA extraction and purification. For both the hypothalamus and placenta, RNA was extracted from tissue/Trizol homogenates via chloroform liquid-liquid extraction and alcohol precipitation. Purification of mRNA was performed using Qiagen RNeasy Mini kits and Illumina cDNA libraries of E18.5 hypothalamus and placental mRNA were prepared from 25 ng and 300 ng total RNA, respectively, using the Illumina Stranded mRNA Prep kits (20040532, Illumina) and indexes (20040553, Illumina) according to the manufacturer's protocol. Library fragment size was quantified using Agilent High Sensitivity D1000 ScreenTape Assays (5067\u0026ndash;5584, Agilent). Library concentrations were determined using the Qubit dsDNA High Sensitivity Assay (Q32851, ThermoFisher). Samples were multiplexed and sequenced on an Illumina NextSeq550 instrument using high output 1x75bp chemistry (20024906, Illumina).\u003c/p\u003e \u003cp\u003eFASTQ files generated from Illumina were concatenated and used as an input to kallisto, a pseudoalignment program, for alignment to the \u003cem\u003eMus musculus\u003c/em\u003e reference transcriptome (version 38) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The remaining analysis took place in the R statistical environment (Version 4.1.1; R Core Team 2021). Gene isoforms were assigned to gene symbols using the Bioconductor package tximport and genes were filtered to counts per million\u0026thinsp;\u0026gt;\u0026thinsp;1 in at least four samples for the hypothalamus and \u0026gt;\u0026thinsp;1 in at least five for placental analysis \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. One wild-type female sample was removed from the hypothalamic analysis due to an issue with library preparation. The filtered gene lists were then normalized using trimmed mean of M-values in edgeR, variance weights were calculated using voom, and differential expression analysis of linear fit models was performed using limma, Benjamini-Hochburg false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.1 and log fold change (logFC)\u0026thinsp;\u0026gt;\u0026thinsp;1 \u003csup\u003e45\u0026ndash;47\u003c/sup\u003e. Although we did not observe significant differences in individual genes based on conservative corrections for multiple comparisons, adjusted p\u0026thinsp;\u0026le;\u0026thinsp;0.05, differential expression of genes with uncorrected p\u0026thinsp;\u0026le;\u0026thinsp;0.05 were assessed as a hypothesis generating approach for future studies.\u003c/p\u003e \u003cp\u003eThe remaining analyses were performed as gene set or cluster-based analysis as we hypothesized that expression of \u003cem\u003eUty\u003c/em\u003e would likely have a significant overall impact on expression of numerous genes and biological processes, as is the function of a GSEA. method. First, heatmaps were generated with all detectable transcripts using unbiased hierarchical clustering of genes based on z-scores for differential gene expression between XX\u0026thinsp;+\u0026thinsp;Uty and XX females (contrast matrix XX\u0026thinsp;+\u0026thinsp;Uty female - XX female) for each tissue. Modules were determined from a row cluster height of 0.3 that resulted in 12 different modules in both the placenta and hypothalamus. The Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics resource was used to analyze gene sets within these modules in order to identify and understand potential biological relevance from broad changes in gene expression associated with \u003cem\u003eUty\u003c/em\u003e expression using GO subsets biological processes (BP), molecular function (MF), and cellular component (CC) and significance threshold set at FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and enrichment score\u0026thinsp;\u0026gt;\u0026thinsp;1 \u003csup\u003e38,39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGene set enrichment analysis (GSEAv4.3.2, Broad Institute, Cambridge, MA) of normalized counts were used to identify patterns of gene expression and determine greater-than-chance enrichment of biological pathways in a threshold-free manner (i.e. without consideration for differential gene expression), as previously reported by our work and that of other labs \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Briefly, collections of GO subcategory biological processes (GO:BP) annotated gene sets were obtained from the Molecular Signature Database (MSigDBv7.4.1, Broad Institute, Cambridge, MA) available for use with GSEA software. Gene set permutations were computed in GSEA to determine FDR, nominal p value, and normalized enrichment score (NES) of each gene set with a significance threshold set at FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and NES\u0026thinsp;\u0026gt;\u0026thinsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypes\u003c/h2\u003e \u003cp\u003eTo communicate the practical significance (i.e., magnitude of effect), effect sizes were calculated for all phenotypic measures. ANOVA effect size was determined by calculating Eta squared (η\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e), effects of which are defined as small at 0.01, medium at 0.06, and large at 0.14. Effect sizes for post-hoc Tukey\u0026rsquo;s or Sidak\u0026rsquo;s corrections for multiple comparisons were calculated by Cohen\u0026rsquo;s d, effects of which are defined as small at 0.2, medium at 0.5, and large at 0.81 \u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Weights\u003c/h2\u003e \u003cp\u003eFor all studies in adult animals, first generation offspring on a B6:129 mixed background received a unique identification ear tag at PN28 and were weighed weekly from PN28 - PN70. At PN70, animals were placed on a high-fat diet (HFD; Research Diets Inc, D12492), calories provided by protein 29% and fat 14%, or remained on the grain-based control chow diet (Purina Rodent Chow 5001, St. Louis, MO), calories provided by protein 20% and fat 60%, to assess phenotypic outcomes following a 5-week dietary challenge. Two-way repeated measures and one-way ANOVAs, with Sidak\u0026rsquo;s or Tukey\u0026rsquo;s multiple comparisons tests, respectively, were conducted using Prism (Graphpad), α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCorticosterone Response to Restraint Stress\u003c/h2\u003e \u003cp\u003ePN56 XX, XY, and XX\u0026thinsp;+\u0026thinsp;Uty offspring underwent restraint stress by being placed in a 50 mL conical tube for 15 min. A single\u0026thinsp;\u0026lt;\u0026thinsp;1mm distal tail snip was made at time 0 to collect 10 \u0026micro;L of tail blood at 0, 15, 30, and 120 minutes, which was placed in EDTA-treated tubes. Quantification of plasma corticosterone levels was determined by radioimmunoassay (207120, MP Biomedicals). Two-way repeated measures ANOVA with Sidak\u0026rsquo;s multiple comparisons post-hoc test was conducted using Prism (Graphpad), α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFood Consumption\u003c/h2\u003e \u003cp\u003eFood consumption was assessed at PN84 for a 24 hr period by weighing food at hour 0 and again at hour 24. Animals were pair-housed in the same genotype pairs for this assessment and data was analyzed as weight of food consumed per animal, assuming each animal ate the same amount of food, and normalized to individual body weight. A one-way ANOVA with Tukey\u0026rsquo;s multiple comparisons post-hoc test was conducted using Prism (Graphpad), α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGlucose Tolerance Test\u003c/h2\u003e \u003cp\u003eA glucose tolerance test was administered at PN91 to XX, XY, and XX\u0026thinsp;+\u0026thinsp;Uty offspring. Animals were fasted for six hours before receiving an intraperitoneal injection of 0.3g/ml glucose in saline. A Contour Next Blood Glucose Monitoring System (Bayer Co, Germany) was used to measure blood glucose levels. A single\u0026thinsp;\u0026lt;\u0026thinsp;1 mm distal tail snip was made to collect tail blood at 0, 30, 60, and 120 min from the time of injection. Two-way repeated measures ANOVA with Sidak\u0026rsquo;s multiple comparisons post-hoc test was conducted using Prism (Graphpad), α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe RNA-sequencing data generated in this study has been deposited in the NCBI SRA database under accession code: \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePRJNA883432\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlacenta and Hypothalamic Gene Expression\u003c/h2\u003e \u003cp\u003eCluster dendrograms and principal components analysis (PCA) showed little separation between XX\u0026thinsp;+\u0026thinsp;Uty and XX females in the placenta (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) or hypothalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Differential gene expression analysis (contrast matrix XX\u0026thinsp;+\u0026thinsp;Uty female - XX female; FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.1, LogFC\u0026thinsp;\u0026gt;\u0026thinsp;1) revealed no significant changes in individual gene expression, with the exception of \u003cem\u003eUty\u003c/em\u003e, between the genotypes in the placenta or hypothalamus. Additionally, expression of \u003cem\u003eUtx\u003c/em\u003e was not altered by overexpression of \u003cem\u003eUty\u003c/em\u003e in XX\u0026thinsp;+\u0026thinsp;Uty animals (\u003cb\u003eSupplemental File 2\u003c/b\u003e). Collectively, these findings support an interpretation that changes in overall gene expression in XX\u0026thinsp;+\u0026thinsp;Uty animals compared to XX did not the result from our random insertion overexpressing transgenic approach. However, as a random insertion method does not allow for confirmation of where or how many insertions in the genome have occurred, we acknowledge this potential methodological confound.\u003c/p\u003e \u003cp\u003eDifferential gene expression analysis was used to generate volcano plots using unadjusted p-values and logFC to identify potential genes of interest associated with Uty-Tg expression. \u003cem\u003eUty\u003c/em\u003e was the only gene that reached statistical significance by adjusted p-value in both the placenta (p\u0026thinsp;=\u0026thinsp;0.001) and hypothalamus (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with increased expression observed in XX\u0026thinsp;+\u0026thinsp;Uty tissues compared to XX females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the placenta and hypothalamus, we identified 618 and 777 genes, respectively, that had unadjusted p\u0026thinsp;\u0026le;\u0026thinsp;0.05. A handful of up- and downregulated genes are highlighted, with one specific gene, \u003cem\u003eCep350\u003c/em\u003e, downregulated in both the placenta and hypothalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHierarchical clustering was performed on all detectable transcripts without p-value or logFC restrictions (p\u0026thinsp;=\u0026thinsp;1, logFC\u0026thinsp;=\u0026thinsp;0) to look at broad differences in gene expression profiles between XX\u0026thinsp;+\u0026thinsp;Uty, XX, and XY animals. In the placenta, there were a total of 14,230 detectable transcripts that were used for hierarchical clustering and grouped into twelve distinct modules. We identified modules with differential gene expression patterns that were directionally similar (i.e., similarly upregulated or downregulated based on z-score) for XX\u0026thinsp;+\u0026thinsp;Uty compared to XX females, similar for XX\u0026thinsp;+\u0026thinsp;Uty females and XY males compared to XX females, as well as modules with differential expression that was unique to XX\u0026thinsp;+\u0026thinsp;Uty (e.g., upregulated in XX\u0026thinsp;+\u0026thinsp;Uty but downregulated in XX and XY), or expression profiles that did not show a change in directionality of regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Modules 4, 5, and 12 are referred to as masculinized because the direction of change in XX\u0026thinsp;+\u0026thinsp;Uty females relative to XX females is the same as the direction of change in XY males relative to XX females. The number of genes in these modules corresponded to 21.3% differential expression XX\u0026thinsp;+\u0026thinsp;Uty and XX, 16.4% differential expression XX\u0026thinsp;+\u0026thinsp;Uty and XY, 49.4% differential expression XX\u0026thinsp;+\u0026thinsp;Uty, and 12.9% without overlapping expression patterns between genotypes. Percentages were based on the total number of detectable transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Enriched biological processes were identified using DAVID functional annotation tools. For analysis, gene lists from modules that showed similarities in directionality of change were grouped together, with modules 2, 7, and 11 as a group and modules 1, 8, and 9 as another group, and input into DAVID separately to assess downregulated (blue) and upregulated (red) biological processes, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and enrichment score\u0026thinsp;\u0026gt;\u0026thinsp;1. Processes enriched in the XX\u0026thinsp;+\u0026thinsp;Uty modules included protein modification, through kinase, phosphatase, transferase, and peptidase enzymes, ribosomal structure and translation, and extracellular matrix assembly. The same approach was used to identify biological processes in the masculinized XX\u0026thinsp;+\u0026thinsp;Uty female gene sets, which showed enrichment for biological processes including kinase and transferase activity, vesicle mediated transport, cholesterol homeostasis and lipid transport, fibrinogen complex, and mRNA processing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Finally, GSEA analysis of normalized counts was performed, with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, NES\u0026thinsp;\u0026gt;\u0026thinsp;0\u0026thinsp;~\u0026thinsp;XX\u0026thinsp;+\u0026thinsp;Uty phenotype, and NES\u0026thinsp;\u0026lt;\u0026thinsp;0\u0026thinsp;~\u0026thinsp;XX female phenotype (\u003cb\u003eSupplemental Data 1\u003c/b\u003e). Dot plots represent the top 20 significantly enriched biological processes that included inflammatory and microbial response pathways, extracellular matrix disassembly, and regulation of peptidase and hydrolase activity ascribed solely to the XX\u0026thinsp;+\u0026thinsp;Uty phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the hypothalamus, the total number of detectable transcripts was 14,553, which were used for hierarchical clustering and grouped into twelve distinct modules. Similar to the placenta, we identified modules with differential gene expression patterns that were directionally similar for XX\u0026thinsp;+\u0026thinsp;Uty compared to XX females, similar for XX\u0026thinsp;+\u0026thinsp;Uty females and XY males compared to XX females, as well as modules with differential expression that was unique to XX\u0026thinsp;+\u0026thinsp;Uty (e.g., upregulated in XX\u0026thinsp;+\u0026thinsp;Uty but downregulated in XX and XY), or expression profiles that did not show a change in directionality of regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Modules 9 and 2 are referred to as masculinized because the direction of change in XX\u0026thinsp;+\u0026thinsp;Uty females relative to XX females is the same as the direction of change in XY males relative to XX females. The number of genes in these modules correspond to 20.2% differential expression XX\u0026thinsp;+\u0026thinsp;Uty and XX, 24.8% differential expression XX\u0026thinsp;+\u0026thinsp;Uty and XY, 36.5% differential expression XX\u0026thinsp;+\u0026thinsp;Uty, and 18.5% without overlapping expression patterns between genotypes. Percentages are based on the total number of detectable transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Enriched biological processes were identified using DAVID as described for the placenta analysis above. Processes enriched in the XX\u0026thinsp;+\u0026thinsp;Uty modules included ion channel activity, microtubule-based movement, cell projection organization, mRNA processing, transferase activity, and regulation of transcription. The same approach was used to identify biological processes in masculinized XX\u0026thinsp;+\u0026thinsp;Uty modules. Biological processes enriched in these modules included synapse and cell junction, transferase activity and ATP-binding, calcium transport, cell cycle and division, and regulation of interferon alpha and beta (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). GSEA was performed as described for the placenta analysis above. In the hypothalamus, biological processes enriched in the XX\u0026thinsp;+\u0026thinsp;Uty phenotype included response to wounding, chromosome segregation, and blood vessel and muscle morphogenesis. However, the XX female phenotype showed enrichment for synaptic signaling, hormone regulation, and feeding behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMasculinized Gene Expression Patterns and Transcriptional Regulation in the XX\u0026thinsp;+\u0026thinsp;Uty Female Placenta and Hypothalamus\u003c/h2\u003e \u003cp\u003eFocusing on the genes that showed male-like differential expression patterns in XX\u0026thinsp;+\u0026thinsp;Uty tissues, or masculinized differential expression patterns, we probed for commonalities between tissues and utilized previously published data sets to assess a role for UTY in sex-specific gene expression and transcriptional regulation. While most of the masculinized differentially expressed genes were not shared between the placenta and hypothalamus, confirming a likely tissue-specific role for UTY, 260 genes did show similar differential expression patterns with 108 upregulated and 152 downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Due to the limited number of genes identified, we were not able to assess enriched biological processes in an upregulated or downregulated specific manner. Instead, DAVID was used to identify enriched biological processes in this list of 260 genes. Enriched processes included ATP-binding and transferase activity, protein phosphorylation, kinase activity, tyrosine activity, and fatty acid oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing previously published datasets reporting transcriptomic sex differences in the placenta and hypothalamus, we compared our masculinized XX\u0026thinsp;+\u0026thinsp;Uty genes to determine if \u003cem\u003eUty\u003c/em\u003e played a broader role in masculinizing gene expression \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. More specifically, we compiled a list of 229 placental genes and 182 hypothalamic genes that showed significant differences, p\u0026thinsp;\u0026le;\u0026thinsp;0.05, in expression between XX females and XY males from these published datasets. In the placenta, we observed a 14% (32 genes) overlap in the placenta and a 21.4% (39 genes) overlap in the hypothalamus between previously reported sex differences in gene expression and masculinized XX\u0026thinsp;+\u0026thinsp;Uty genes (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Of interest and developmentally important, we identified estrogen receptor alpha (\u003cem\u003eEsr1\u003c/em\u003e) and several estrogen responsive genes, oxytocin (\u003cem\u003eOxt\u003c/em\u003e), insulin-like growth factor 1 (\u003cem\u003eIgf1\u003c/em\u003e), and protein kinase C delta (\u003cem\u003ePrkcd\u003c/em\u003e), in both our masculinized XX\u0026thinsp;+\u0026thinsp;Uty gene list and reported sex differences in the hypothalamus, suggesting a potential interaction between UTY and estrogen receptor transcriptional regulation \u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53 CR54\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe ability of UTY to function as a demethylase, similar to its X-linked homologue, UTX, has yet to be determined. We integrated our previously published H3K27me3 chromatin immunoprecipitation sequencing (ChIP-Seq) dataset from the placenta of mice on a B6:129 background with our masculinized XX\u0026thinsp;+\u0026thinsp;Uty female placental genes \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To test the hypothesis that the presence of UTX and UTY in male (XY) cells would contribute to increased H3K27me3 demethylation and therefore fewer H3K27me3 marks at transcriptional start sites, we first filtered the ChIP-Seq data accordingly. This process involved removing duplicate transcriptional start sites (TSS) from the ChIP-Seq dataset, identifying genes with reduced TSS counts in males (XY) compared to females (XX), and then comparing the list of genes to our masculinized XX\u0026thinsp;+\u0026thinsp;Uty gene list that included both upregulated and downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). This process revealed 1155 genes shared between the filtered ChIP-Seq dataset and the masculinized XX\u0026thinsp;+\u0026thinsp;Uty gene list, resulting in 9.5% of all genes we had previously identified with H3K27me3 TSS counts where XY\u0026thinsp;\u0026lt;\u0026thinsp;XX and 49.5% of the masculinized XX\u0026thinsp;+\u0026thinsp;Uty gene list (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The biological processes enriched in the 1155 genes include transferase and kinase activity, nucleotide and ATP-binding, and lipid transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eUty Confers Vulnerability to Metabolic Dysregulation in HFD Females\u003c/h2\u003e \u003cp\u003eA significant effect of genotype was observed for baseline body weight measurements (F (2,29)\u0026thinsp;=\u0026thinsp;63.4, p\u0026thinsp;=\u0026thinsp;0.0001; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.81; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), with males consistently heavier than XX\u0026thinsp;+\u0026thinsp;Uty (PN28, p\u0026thinsp;=\u0026thinsp;0.02; PN35 \u0026ndash; PN70, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;1.98) and XX females (PN35, p\u0026thinsp;=\u0026thinsp;0.0002; PN42\u0026ndash;70, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;1.81). No significant effect of Uty-Tg was observed for baseline female body weight measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). No main effect of genotype was observed for stress response measured by restraint stress and HPA activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), however post-hoc analysis did reveal that XY males had significantly lower levels of corticosterone at 120 minutes compared to XX and XX\u0026thinsp;+\u0026thinsp;Uty females (p\u0026thinsp;=\u0026thinsp;0.007; d\u0026thinsp;=\u0026thinsp;0.65; p\u0026thinsp;=\u0026thinsp;0.04; d\u0026thinsp;=\u0026thinsp;0.56). When animals were challenged with a HFD, a significant effect of genotype was observed (F (2,29\u0026thinsp;=\u0026thinsp;85.08, p\u0026thinsp;=\u0026thinsp;0.0001; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.85; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), where males were heavier than XX\u0026thinsp;+\u0026thinsp;Uty (Wk1\u0026ndash;5, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;3.70; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and XX females (Wk1\u0026ndash;5, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;2.86; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Body weight assessments at the end of the 5 week dietary challenge revealed a significant effect of genotype (F(2,29)\u0026thinsp;=\u0026thinsp;79.50, p\u0026thinsp;=\u0026thinsp;0.0001; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.85; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), with XX females weighing less than XY males (p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;4.52; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and XX\u0026thinsp;+\u0026thinsp;Uty females weighing less than XY males and XX females (p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;5.50 and p\u0026thinsp;=\u0026thinsp;0.03; d\u0026thinsp;=\u0026thinsp;1.08; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). A significant effect of genotype was observed for percent weight gained on HFD over the five-week period (F (2,29)\u0026thinsp;=\u0026thinsp;10.45, p\u0026thinsp;=\u0026thinsp;0.0004; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.42; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), with XX\u0026thinsp;+\u0026thinsp;Uty females consistently having a lower percent weight gained than XY males (Wk2, p\u0026thinsp;=\u0026thinsp;0.01; Wk3, p\u0026thinsp;=\u0026thinsp;0.0002; Wk4, p\u0026thinsp;=\u0026thinsp;0.003; Wk5, p\u0026thinsp;=\u0026thinsp;0.0004; d\u0026thinsp;=\u0026thinsp;1.50; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This significant effect of genotype persisted to the end of the five-week dietary challenge (F (2,29)\u0026thinsp;=\u0026thinsp;12.65, p\u0026thinsp;=\u0026thinsp;0.0001; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.47; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), where XX\u0026thinsp;+\u0026thinsp;Uty females had a significantly lower percent weight change than XY males and XX females (p\u0026thinsp;=\u0026thinsp;0.02; d\u0026thinsp;=\u0026thinsp;2.54; p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;1.14; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). To assess if these changes in body weight corresponded to changes in glucose regulation and food consumption, we ran a glucose tolerance test (GTT) and measured the 24-hour food consumption. Genotype had a significant effect on glucose tolerance (F (2,31)\u0026thinsp;=\u0026thinsp;4.407, p\u0026thinsp;=\u0026thinsp;0.02; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.22; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), with XX\u0026thinsp;+\u0026thinsp;Uty females having significantly lower glucose levels at 60 and 120 minutes compared to XY males (p\u0026thinsp;=\u0026thinsp;0.0002; p\u0026thinsp;\u0026le;\u0026thinsp;0.0001; d\u0026thinsp;=\u0026thinsp;0.48; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Finally, a significant effect of genotype was observed for 24-hr food consumption (F(2,11)\u0026thinsp;=\u0026thinsp;11.87, p\u0026thinsp;=\u0026thinsp;0.0018; η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.68) with XY males consuming significantly more food compared to XX (p\u0026thinsp;=\u0026thinsp;0.02; d\u0026thinsp;=\u0026thinsp;2.08) and XX\u0026thinsp;+\u0026thinsp;Uty females (p\u0026thinsp;=\u0026thinsp;0.0014; d\u0026thinsp;=\u0026thinsp;4.89; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSex differences in fetal development and sensitivity to intrauterine perturbations are well documented, however, there remains a paucity of information on specific molecular and/or genetic mechanisms responsible for divergent male and female development and health outcomes. We previously showed that OGT, a protein encoded by an X-linked gene, contributes to female fetal protection from prenatal stress, in part through its regulation of the H3K27 methyltransferase, EZH2 \u003csup\u003e31,32,34\u003c/sup\u003e. Increased OGT is associated with higher levels of H3K27me3 in the female placenta, in both mouse and human tissue \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, less is understood as to the sex-specific roles of the X- and Y-linked H3K27 demethylases, UTX and UTY. Comparisons of UTX and UTY amino acid sequences demonstrate extensive sequence homology, reaching 88% homology, both within and outside of the catalytic domain, yet the function of UTY remains less clear \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Important to sex-differences in H3K27me3-mediated transcriptional regulation in the placenta, XY tissues naturally have reduced OGT in addition to the presence of UTY, potentially culminating in overall reduced levels of H3K27me3 and reduced transcriptional repressive control in tissues such as the placenta \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. As such, we hypothesized that these differences may account for an increased vulnerability for males in utero, especially for neurodevelopmental risk \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We previously reported no sex difference for placental \u003cem\u003eUtx\u003c/em\u003e expression suggesting that \u003cem\u003eUtx\u003c/em\u003e, unlike \u003cem\u003eOgt\u003c/em\u003e, may not escape X inactivation in the placenta \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that if UTY indeed had transcriptional influence, either via potential demethylation of H3K27me3 and/or other as of yet unidentified activity, it would serve as a molecular mechanism that could direct male-specific fetal development. Therefore, we generated a \u003cem\u003eUty\u003c/em\u003e-overexpressing transgenic mouse and utilized a transcriptomic approach to assess potential changes in placental and fetal brain development.\u003c/p\u003e \u003cp\u003eInitial phenotypic assessment found no overt developmental changes or outcomes in the XX\u0026thinsp;+\u0026thinsp;Uty mice. As \u003cem\u003eUty\u003c/em\u003e is not a master regulator (i.e., not known to be at the top of a gene regulation hierarchy) and limited protein-protein interactions have been described for UTY, we did not anticipate that over-expression of \u003cem\u003eUty\u003c/em\u003e in a wild-type female mouse would produce profound phenotypic changes. We examined global health outcomes in the XX\u0026thinsp;+\u0026thinsp;Uty mice and confirmed that they showed no signs of developmental abnormalities or health consequences resulting from \u003cem\u003eUty\u003c/em\u003e expression. Significant differences in gene expression based on conservative corrections for multiple comparisons and distinct differences in clustering assessed by cluster dendrogram and principal components analysis were not observed in the placenta or hypothalamus. We interpret these findings as an indication that we have not caused significant, potentially detrimental changes to the genome by the random insertion approach. Furthermore, the lack of baseline changes in body weight and functionality of the stress-axis (HPA) in XX\u0026thinsp;+\u0026thinsp;Uty compared to XX animals indicates that growth, metabolism, and neuroendocrine regulation were relatively normal, under control housing and feeding conditions.\u003c/p\u003e \u003cp\u003eUtilizing our transcriptomic results as a hypothesis generating dataset, we adjusted our statistical parameters to examine relationships and patterns based on unadjusted p-values and in all detectable transcripts from our list of differentially expressed genes. We first compared gene sets between tissues to identify those that were similarly regulated in both. In the placenta and developing hypothalamus of XX\u0026thinsp;+\u0026thinsp;Uty animals, we found 206 genes that were enriched for biological processes including autophosphorylation and kinase activity. Autophosphorylation of protein kinases is a common process by which the enzyme adds a phosphate group to itself, altering its catalytic activity. Examples of such tyrosine kinases include epidermal growth factor receptor, insulin receptors, and SRC kinases that are important for proliferation, differentiation, and metabolism \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In addition to the shared biological processes, we also identified the gene for centrosome-associated protein 350 (\u003cem\u003eCep350\u003c/em\u003e) as a similarly downregulated gene in XX\u0026thinsp;+\u0026thinsp;Uty placenta and hypothalamus. CEP350 is required for the anchoring of microtubules and recruitment of some nuclear receptors, such as peroxisome proliferator-activated receptor alpha (PPARα), a major regulator of lipid metabolism. Centrosomal genes, including \u003cem\u003eCep350\u003c/em\u003e, play a pivotal structural role in primary cilia that are antenna-like sensory organelles found in the placenta and hypothalamus to coordinate developmental processes and metabolic homeostasis \u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Furthermore, defective cilia have been implicated in the pathogenesis of preeclampsia and metabolic disorders \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Dysregulation of \u003cem\u003eCep350\u003c/em\u003e associated with \u003cem\u003eUty\u003c/em\u003e expression likely deserves further interrogation as a male-specific source of vulnerability for pregnancy complications and metabolic dysregulation. These overlapping gene sets in the placenta and hypothalamus of XX\u0026thinsp;+\u0026thinsp;Uty animals consistently suggest an association between \u003cem\u003eUty\u003c/em\u003e expression and regulation of genes important for metabolism.\u003c/p\u003e \u003cp\u003eUnique to the placenta, almost 50% of the 14,230 detected transcripts in the placenta showed differential expression profiles that were completely distinct to XX\u0026thinsp;+\u0026thinsp;Uty animals. Gene ontology (GO) identified biological processes related to transcription, translation, and post-translational protein modifications, suggesting that UTY plays a regulatory role in gene expression and protein synthesis and modification. Not surprisingly, we saw a\u0026thinsp;~\u0026thinsp;20% overlap in gene regulation between XX\u0026thinsp;+\u0026thinsp;Uty and XX females, but we focused on the potential masculinizing effects of UTY on gene regulation, (i.e., pattern similarities between XX\u0026thinsp;+\u0026thinsp;Uty and XY mice), as evidence of novel UTY function. We found that 16.4% of detectable transcripts were male-biased in their expression pattern in the placenta where biological processes vital to cellular signaling, metabolism, and inflammation were enriched, including protein ubiquitination \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUbiquitination is a common post-translational protein modification that tags proteins for sorting, localization, trafficking, and degradation. Therefore, protein ubiquitination is essential to both intracellular and extracellular communication that can impact diverse cellular processes such as DNA transcription, cell cycle, ribosome biogenesis, and inflammation. For example, new evidence suggests that ubiquitinated proteins are directed toward extracellular secretion via membrane-bound vesicles known as extracellular vesicles (EVs). EVs serve as carriers for a variety of proteins, microRNAs, mRNAs, and lipids transporting these biological molecules to neighboring cells and mediating physiological processes involved in development and homeostasis \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. While the physiological consequences of altered protein ubiquitination associated with \u003cem\u003eUty\u003c/em\u003e expression are impossible to discern without further studies, it may be one underlying mechanism that contributes to several other biological processes enriched in our masculinized XX\u0026thinsp;+\u0026thinsp;Uty female dataset, including vesicle transport, cholesterol homeostasis, and inflammation.\u003c/p\u003e \u003cp\u003eRegulation of immune tolerance is essential for successful pregnancy and relies on coordination and communication between neighboring cells. The semi-allogeneic placenta derives half of its genetic material from the mother and half from the father which leads to the expression of foreign, paternal, proteins in the intrauterine environment \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Therefore, several mechanisms are in place to protect the placenta from being targeted by the maternal immune system \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. We observed alterations in the regulation of pathways associated with inflammatory and immune responses in placentas with \u003cem\u003eUty\u003c/em\u003e expression that raises some questions regarding the role of UTY in chronic placental inflammation, a phenotype more often observed with male fetuses and associated with premature delivery \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Other studies have reported associations between experimentally reduced \u003cem\u003eUty\u003c/em\u003e expression and changes in immune response, although there are inconsistencies in the directionality of immune dysregulation \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDisruption of cholesterol homeostasis also deserves further interrogation as a pathway that is partially regulated by UTY in the placenta. The placenta acquires cholesterol from maternal circulation and synthesizes cholesterol that is subsequently transferred to the fetus or used by the placenta to make hormones. Cholesterol levels in healthy pregnancies increase over time. However, too much cholesterol is associated with increases in lipid peroxidation, reactive oxygen species, and inflammation, as well as pregnancy complications including preeclampsia and fetal growth restriction \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. These similarly regulated pathways between XX\u0026thinsp;+\u0026thinsp;Uty and XY placentas provide novel evidence that UTY might play a role in male-specific vulnerability to inflammation and pregnancy complications \u003cem\u003ein utero\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eUnique to the hypothalamus, 14,553 transcripts were detected. Compared to the placenta, there were fewer genes distinct to the XX\u0026thinsp;+\u0026thinsp;Uty females (36.5%), and more genes with similar expression profiles between XX\u0026thinsp;+\u0026thinsp;Uty and XY (24.8%). We also detected a greater number of genes (39) overlap between the masculinized XX\u0026thinsp;+\u0026thinsp;Uty gene list and previously published male-biased genes in the developing mouse hypothalamus, including estrogen receptor alpha (\u003cem\u003eEsr1\u003c/em\u003e), and several estrogen responsive genes, insulin-like growth factor 1 (\u003cem\u003eIgf1\u003c/em\u003e), oxytocin (\u003cem\u003eOxt\u003c/em\u003e), and protein kinase C delta (\u003cem\u003ePrkcd\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53 CR54\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In the developing brain, testosterone aromatized to estradiol masculinizes the organization of neural circuits \u003csup\u003e\u003cspan additionalcitationids=\"CR72 CR73 CR74 CR75\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Thus, it is interesting to suggest that UTY may have a more \u0026lsquo;masculinizing\u0026rsquo; effect on the hypothalamic transcriptome compared to the placenta. However, the biological pathways enriched in both the XX\u0026thinsp;+\u0026thinsp;Uty unique gene list and masculinized XX\u0026thinsp;+\u0026thinsp;Uty gene list were less specialized in the brain and more broadly related to the regulation of gene expression, protein phosphorylation, and RNA splicing and processing that may reflect cell type specific and/or timing of analysis outcomes. Our findings, along with recently reported evidence that UTY plays a role in male-specific neural stem cell differentiation, demonstrate that UTY may be a contributing molecular mechanism by which sex differences in brain organization arise \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe capacity for UTY to function as an H3K27me3 demethylase, like its X-linked homologue UTX, has been scrutinized for decades \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Utilizing previously published data from our lab, we integrated corresponding genes that were identified by H3K27me3 ChIP-Seq with our masculinized XX\u0026thinsp;+\u0026thinsp;Uty placenta dataset \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. More specifically, we limited the placental ChIP-Seq data set so that we were only examining genes with transcriptional start sites that had counts lower in males compared to XX females, with the assumption that these genes were demethylated in a more male-specific direction. Interestingly, a small percentage of the genes from the masculinized XX\u0026thinsp;+\u0026thinsp;Uty group (9.5%) overlapped with the filtered ChIP-Seq gene list, however, that small percentage accounted for nearly 50% of the total masculinized XX\u0026thinsp;+\u0026thinsp;Uty genes. From these data we are not able to determine whether UTY may participate in demethylation of H3K27me3 in a direct or indirect manner, however, these findings suggest that a substantial portion of the masculinized XX\u0026thinsp;+\u0026thinsp;Uty genes have a reduction in this transcriptional repressive mark in the presence of UTY.\u003c/p\u003e \u003cp\u003eIn examination of adult phenotypes for effects of UTY, while adult XX\u0026thinsp;+\u0026thinsp;Uty females showed no significant differences from XX females under normal control conditions, they presented with surprising alterations in body weight and glucose tolerance following a 5-week calorically dense dietary challenge. While we hypothesized that a detectable phenotype would resemble wild-type males, we observed that XX\u0026thinsp;+\u0026thinsp;Uty females did not present in a masculinized manner. Surprisingly, XX\u0026thinsp;+\u0026thinsp;Uty females at the end of the HFD exposure weighed less and had a \u003cem\u003egreater\u003c/em\u003e glucose clearance compared to either wild-type males or females. As this phenotype was unique to XX\u0026thinsp;+\u0026thinsp;Uty females, we interpret this finding as a potential role for UTY in metabolic regulation that may be related to developmental changes or current adult activity of UTY and may be distinct from XY and XX mice as a result of conflicting or compounding effects of UTY on an XX background. In examination of food consumption as an explanation for body weight differences, we found that XX\u0026thinsp;+\u0026thinsp;Uty females in fact consumed fewer calories than males on the HFD. However, no significant differences were detected between XX\u0026thinsp;+\u0026thinsp;Uty females and XX females suggesting again unique interactions of UTY on an XX background that are impacting metabolic processes. Future investigations into the mechanisms and hormonal regulation driving this \u003cem\u003eUty\u003c/em\u003e-associated phenotype could provide important insight about sex differences in metabolism and risk of metabolic disorders.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eEpigenetic regulators expressed from X and Y chromosomes have significant potential to impart broad sex differences in transcriptional regulation, and to contribute to sex-specific responses to environmental perturbations. In such studies, the functional role of UTY has largely been ignored, however, the few studies that do exist suggest that it retains biological functions that may be independent from H3K27me3 demethylation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The data reported herein suggest that UTY does indeed play a role in male-specific transcription. We specifically focused on the placenta and hypothalamus for this study due to the plethora of evidence showing that these tissues respond to changes in the intrauterine environment in a sex-specific manner. While our transcriptomic analysis was tissue-specific, our expression of \u003cem\u003eUty\u003c/em\u003e was not. Therefore, an important limitation of our study is regarding the global overexpression of \u003cem\u003eUty\u003c/em\u003e and the broad changes in any tissue that could produce secondary effects in the placenta or brain, and vice versa. Moving forward it would be important to modify the \u003cem\u003eUty\u003c/em\u003e construct to conditionally express the transgene. Furthermore, the random insertion of transgenes rather than a targeted insertion approach can disrupt normal gene sequences and yield unpredictable phenotypes and is a potential confounding factor in our study. We recognize the importance of utilizing site-specific overexpression and knockout models in future studies to probe if deletion of \u003cem\u003eUty\u003c/em\u003e results in loss of a proposed functions in XY males. More work is needed to further characterize the molecular functions and contributions to phenotypic outcomes by UTY. In conclusion, these studies provide novel insight as to the potential roles of UTY in male-specific gene expression that may underlie sex differences in risk and resilience across the lifespan and provide a foundation to build on in future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch in this manuscript was supported by the National Institute of Child Health and Human Development under award number R01HD097093 (T.L.B). The published content is the responsibility of the authors and does not necessarily represent the views of the National Institutes of Health. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization (TB, BN), experimental design (TB, KR, LF, HZ), experimental execution (KR, LF, HZ, RMR, BN, NAL), drafted the manuscript (KR), edited the manuscript (TB, LF, HZ, RMR, BN, KR). \u0026nbsp;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq data generated in this study has been deposited in the NCBI SRA database under accession code PRJNA883432\u003cu\u003e.\u0026nbsp;\u003c/u\u003eAll other raw data are available upon request by contacting the corresponding author, Dr. Tracy Bale.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Gen-H, Genetic Recombineering Heidelberg, for producing and validating the Uty-Tg construct. We would also like to thank Dr. Daniel Beiting at the University of Pennsylvania for access to the base r script through his DIY.transcriptomics course that was used for RNA-sequencing analysis and Dr. Chris Lengner for his assistance in developing the \u003cem\u003eUty\u003c/em\u003e transgenic mouse. Finally, we thank Dr. Eldin Ja\u0026scaron;arević and Dr. Kathleen E. Morrison for their insight and guidance with RNA-sequencing sample preparation and data analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRegitz‐Zagrosek, V. Sex and gender differences in health: Science \u0026amp; Society Series on Sex and Science. \u003cem\u003eEMBO Rep.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 596\u0026ndash;603 (2012).\u003c/li\u003e\n\u003cli\u003eBramble, M. S., Lipson, A., Vashist, N. \u0026amp; Vilain, E. 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Chem.\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 18302\u0026ndash;18313 (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Uty, Kdm6c, Sex Differences, Transcriptional Regulation, Placenta, Hypothalamus, Development, Metabolism","lastPublishedDoi":"10.21203/rs.3.rs-2928137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2928137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe genetic material encoded on X and Y chromosomes provides the foundation by which biological sex differences are established. Epigenetic regulators expressed on these sex chromosomes, including \u003cem\u003eKdm6a\u003c/em\u003e (\u003cem\u003eUtx\u003c/em\u003e), \u003cem\u003eKdm5c\u003c/em\u003e, and \u003cem\u003eDdx3x\u003c/em\u003e have far-reaching impacts on transcriptional control of phenotypic sex differences. Although the functionality of UTY \u003cem\u003e(Kdm6c\u003c/em\u003e, the Y-linked homologue of UTX), has been supported by more recent studies, its role in developmental sex differences is not understood. Here we test the hypothesis that UTY is an important transcriptional regulator during development that could contribute to sex-specific phenotypes and disease risks across the lifespan. We generated a random insertion \u003cem\u003eUty\u003c/em\u003e transgenic mouse (Uty-Tg) to overexpress \u003cem\u003eUty\u003c/em\u003e. By comparing transcriptomic profiles in developmental tissues, placenta and hypothalamus, we assessed potential UTY functional activity, comparing \u003cem\u003eUty\u003c/em\u003e-expressing female mice (XX\u0026thinsp;+\u0026thinsp;Uty) with wild-type male (XY) and female (XX) mice. To determine if \u003cem\u003eUty\u003c/em\u003e expression altered physiological or behavioral outcomes, adult mice were phenotypically examined. \u003cem\u003eUty\u003c/em\u003e expression masculinized female gene expression patterns in both the placenta and hypothalamus. Gene ontology (GO) and gene set enrichment analysis (GSEA) consistently identified pathways focused on transcriptional regulation, immune response, and lipid homeostasis as biological processes associated with UTY. Interestingly, adult females expressing \u003cem\u003eUty\u003c/em\u003e gained less weight and had a greater glucose tolerance compared to wild-type male and female mice when provided a high-fat diet. Utilizing a \u003cem\u003eUty\u003c/em\u003e-overexpressing transgenic mouse, our results provide novel evidence as to a functional transcriptional role for UTY in developing tissues, and a foundation to build on its prospective capacity to influence sex-specific developmental and health outcomes.\u003c/p\u003e","manuscriptTitle":"Developmental sex-specific transcriptomic patterns can be altered by transgenic expression of Uty","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-22 14:55:39","doi":"10.21203/rs.3.rs-2928137/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-06-13T10:55:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-06-03T04:32:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6211beff-db96-4806-85ab-b5c5d5f05d7e","date":"2023-05-27T16:30:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-26T14:28:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-26T13:53:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-05-18T11:14:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-05-18T11:08:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-05-12T14:28:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5f53ae37-9c78-4df8-9c3b-8817fa458729","owner":[],"postedDate":"May 22nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":21619845,"name":"Biological sciences/Developmental biology"},{"id":21619846,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2023-12-04T15:03:12+00:00","versionOfRecord":{"articleIdentity":"rs-2928137","link":"https://doi.org/10.1038/s41598-023-47977-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-11-30 15:01:01","publishedOnDateReadable":"November 30th, 2023"},"versionCreatedAt":"2023-05-22 14:55:39","video":"","vorDoi":"10.1038/s41598-023-47977-x","vorDoiUrl":"https://doi.org/10.1038/s41598-023-47977-x","workflowStages":[]},"version":"v1","identity":"rs-2928137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2928137","identity":"rs-2928137","version":["v1"]},"buildId":"re_ckhLnmML6MCF96OHNJ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}