{"paper_id":"28c1d138-7e58-4757-ac79-d8f40f101b2f","body_text":"PROSER1 Modulates DNA Demethylation through Dual Mechanisms to Prevent Syndromic 1 \nDevelopmental Malformations 2 \n  3 \nAnna Fleming1, Elena V. Knatko1, Xiang Li1, Ansgar Zoch2,3,8, Zoe Heckhausen4,5, Stephanie Stransky6, 4 \nAlejandro J. Brenes7, Simone Sidoli6, Petra Hajkova4,5, Dónal O’Carroll2,3, Kasper D. Rasmussen1 5 \n 6 \n1Division of Molecular, Cellular, and Developmental Biology, University of Dundee, Dundee, UK 7 \n2Centre for Regenerative Medicine, Institute for Regeneration and Repair, Institute for Stem Cell 8 \nResearch, University of Edinburgh, 5 Little France Drive, Edinburgh, EH16 4UU, UK. 9 \n3Wellcome Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born 10 \nCrescent, Edinburgh, EH9 3BF, UK. 11 \n4MRC Laboratory of Medical Sciences, London, UK 12 \n5Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK 13 \n6Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY , USA 14 \n7Division of Cell Signalling and Immunology, University of Dundee, Dundee, UK 15 \n8present address: MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, 16 \nWestern General Hospital, Crewe Road South, Edinburgh EH4 2XU, Edinburgh, UK 17 \n 18 \nCorresponding author: kdrasmussen@dundee.ac.uk 19 \n 20 \nRunning head: PROSER1 in TET function in development 21 \nKeywords: TET1, TET2, TET3, DNA methylation, TOPD, Development, Neurodevelopmental disorder.  22 \n 23 \n  24 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nABSTRACT 25 \nThe link between DNA methylation and neurodevelopmental disorders is well established. However, 26 \nhow DNA methylation is fine-tuned – ensuring precise gene expression and developmental fidelity – 27 \nremains poorly understood. PROSER1, a known  TET2 interact or, was recently linked to a severe  28 \nneurodevelopmental disorder. Here, we demonstrate that PROSER1 interacts with all TET enzymes and 29 \nstabilizes chromatin -bound TET-OGT-PROSER1-DBHS (TOPD) complexes, which regulate DNA 30 \ndemethylation and developmental gene expression. Surprisingly, we find that PROSER1 also 31 \nsequesters TET enzymes, preventing widespread demethylation and transposable element de-32 \nrepression. Our findings identify PROSER1 as a key factor which both positively and negatively 33 \nregulates DNA demethylation essential for mammalian neurodevelopment. 34 \n 35 \nINTRODUCTION  36 \nDNA methylation is a fundamental epigenetic process that is essential for normal development. 37 \nCollaborating with other chromatin -based epigenetic mechanisms, it safeguards the genome by 38 \nsilencing transposable elements, restricts imprinted gene expression, and balanc es gene dosage 39 \nbetween sexes (Jones 2012) . Furthermore, DNA methylation  can in some cases  directly influence 40 \ntranscription factor binding and gene expression through epigenetic modification of promoter and 41 \nenhancer regions (Schübeler 2015; Rasmussen et al. 2015, 2019; Kreibich et al. 2023). The TET family 42 \nof DNA demethylases consists of three members (TET1, TET2, and TET3). These closely related proteins 43 \ncontain a conserved catalytic domain that can iteratively oxidize 5 -methylcytosine (5mC) to 5 -44 \nhydroxymethylcytosine (5hmC), 5 -formylcytosine, and 5 -carboxycytosine, and promote DNA 45 \ndemethylation (Rasmussen and Helin 2016). Mice lacking all three TET enzymes are unable to survive 46 \nbeyond the early stages of development due to gastrulation failures (Dai et al. 2016), while embryos 47 \nlacking only TET3 can progress to the neonatal stage (Gu et al. 2011). Similarly, although mice lacking 48 \neither TET1 or TET2 develop normally (Dawlaty et al. 2011)(Moran-Crusio et al. 2011; Quivoron et al. 49 \n2011)(Li et al. 2011) (Ko et al. 2011) , the combined loss of these enzymes causes developmental 50 \nabnormalities and increased mortality in a proportion of newborn mice  (Dawlaty et al. 2013) . The 51 \nspectrum of developmental defects in knockout mouse lines indicates  that TET enzymes have both 52 \nunique and redundant roles in maintaining developmental processes during early embryonic 53 \ndevelopment.    54 \nThe function of TET enzymes is modulated via protein -protein interactions with a diverse set 55 \nof binding partners. For instance, TET enzymes interact with O-linked N-acetylglucosamine (O-GlcNAc) 56 \ntransferase (OGT) (Chen et al. 2013; Vella et al. 2013; Deplus et al. 2013) and the SIN3A-HDAC histone 57 \ndeacetylase complex (Zhang et al. 2015; Williams et al. 2011; Flores et al. 2023; Zhu et al. 2018)  to 58 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\npromote histone O-GlcNAcylation and histone deacetylation, respectively. In addition, TET1 and TET2 59 \ninteract with the two Drosophila behaviour/human splicing (DBHS) proteins Paraspeckle component 1 60 \n(PSPC1) and Non -POU Domain Containing Octamer Binding (NONO)  (Knott et al. 2016, 2022)  to 61 \nmodulate expression of endogenous retroviruses and bivalent genes (Guallar et al. 2018; Huang et al. 62 \n2022; Li et al. 2020). Finally, TET2 has recently been reported to interact with Proline and Serine Rich 63 \n1 (PROSER1) in the context of UTX and the MLL3/4 branch of COMPASS (Complex of proteins 64 \nassociated with SET1) to modulate H3K4me1 and H3K4me2 levels at UTX binding sites in the human 65 \nembryonic kidney cell line HEK293 (Wang et al. 2021). The relative importance of these interactions in 66 \nregulating TET activity and function is an active area of research. 67 \nInterestingly, recent findings have linked homozygous loss-of-function mutations in PROSER1 68 \nto a novel developmental disorder . This condition features  hypotonia, developmental delays, 69 \ngenitourinary malformations, and craniofacial abnormalities  associated with sensorineural hearing 70 \nloss and strabismus (Salah et al. 2022) . Given the established role of DNA methylation in 71 \nneurodevelopment, as evidenced by the association of mutations in DNMT1, DNMT3A, DNMT3B, 72 \nUSP7, and TET3 with a wide spectrum of developmental disorders (Nava and Arboleda 2024) , the 73 \nobserved interaction between PROSER1 and TET2 is intriguing. While this interaction suggests a 74 \npotential mechanism underlying PROSER1 -associated syndromes, the precise pathological 75 \nconsequences of PROSER1 deficiency on DNA methylation, gene expression, and  ultimately 76 \ndevelopmental integrity during early embryogenesis remain to be elucidated. 77 \n 78 \nRESULTS AND DISCUSSION  79 \nLoss of PROSER1 increases preweaning lethality and is associated with developmental disabiliti es 80 \nand craniofacial abnormalities 81 \nTo establish a direct causal link between PROSER1 loss -of-function gene mutations and 82 \nneurodevelopmental disorders, w e generated a mouse line with constitutive inactivation of the 83 \nendogenous Proser1 gene using CRISPR-Cas9 gene editing (Fig. S1A, B, C). Proser1+/- mice were viable 84 \nand fertile and these mice were, upon backcrossing to C57BL/6J, intercrossed to generate mice with 85 \nhomozygous PROSER1 loss. Analysis of offspring from these breeders demonstrated that P ROSER1 86 \nknockout results in partially penetrant pre-weaning lethality (Fig. 1A). We did not observe prominent 87 \nincreases in perinatal lethality suggesting that most PROSER1 knockout embryos may be reabsorbed 88 \nin utero  during early gestation. Surviving P ROSER1 knockout animals weighed less upon reaching 89 \nadulthood (Fig. 1B) and displayed frequent eye abnormalities including microphthalmia, 90 \nanophthalmia, and cataracts as well as intermittent tremors  and failure-to-thrive (Fig. 1C, 1D ) . To 91 \nfurther characterise neuroanatomical defects, we performed microcomputed tomography (microCT) 92 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nscanning of skulls from adult animals  (8-14 weeks old) . Consistent with reduced overall weight, 93 \nvolumetric analysis revealed reduced cranial bone volume in PROSER1 knockout animals compared to 94 \nwildtype littermates  (Fig. 1E) , while bone density remained unchanged  (Fig. 1F) . Of note, the 95 \npersistence of this phenotype  in fully grown animals  suggests that loss of P ROSER1 result s in 96 \npermanent developmental disability rather than developmental delays. Further comparison of skull 97 \nshape revealed distinct malformations of the maxillary and frontal bones as well as a tendency for 98 \nrounded heads and a  general shortening of the skull (Fig. 1G) . In summary, germline PROSER1 99 \ndeficiency in mice results in pleiotropic developmental abnormalities that resemble the human 100 \nneurodevelopmental disorder in which PROSER1 is mutated. 101 \n   102 \nPROSER1 is a pan-TET interactor and participates in chromatin-associated TOPD complexes  103 \nOur data demonstrate functional homology between mouse and human PROSER1, suggesting that 104 \nmouse embryonic stem cells (mESCs)  and their differentiation can serve as an appropriate and 105 \ntractable model system to elucidate the mechanistic role of PROSER1 in early development. A previous 106 \nstudy reported a protein-protein interaction between PROSER1 and TET2 in HEK293 cells (Wang et al. 107 \n2021). To determine the conservation of this interaction in mESCs and its potential extension to the 108 \nentire TET family of enzymes, we raised two specific anti -murine PROSER1 antibodies with epitopes 109 \nfrom PROSER1 C - and N -termini, respectively, and performed endogenous IP -MS using PROSER1 110 \nknockout mESCs as background control (Fig. 2A, S1D, E, F). Analysis of biological triplicate experiments 111 \nidentified significant enrichment of TET1 and TET2 as well the previously identified TET protein 112 \ninteractors OGT, PSPC1 and NONO. These interactions were also observed in protein lysates isolated 113 \nfrom mESC-derived embryoid bodies (EBs). Unlike mESCs, EBs express TET3 and indeed we observed 114 \nrobust enrichment of all three TET enzymes upon PROSER1 IP (Fig. 2B).  Importantly, by performing 115 \nTET2 IP -MS in wildtype and TET2 knockout mESCs we could furthermore recover PROSER1, OGT, 116 \nPSPC1, and NONO interactions, but no detectable interaction with the other TET enzymes  (Fig. 2C). 117 \nThese findings indicate that the presence of TET2 in PROSER1 -containing complexes is mutually 118 \nexclusive with TET1  and TET3, suggesting that PROSER1 forms discrete complexes with each TET  119 \nprotein. Indeed, PROSER1 IP -MS in TET2 knockout cells still robustly enriched TET1 and TET3, 120 \ndemonstrating that their interaction with PROSER1 is not dependent on TET2 (Fig. S2A). 121 \nOur data suggest the existence of multiprotein complexes involving TET proteins, OGT, and 122 \nPROSER1, as well as members of the DBHS family, which are hereafter referred to as TOPD (TET-OGT-123 \nPROSER1-DBHS proteins) complexes (Fig. 2D). The relative abundance of these complexes is likely to 124 \nbe affected by variation in the expression of TET proteins and their interactors in different tissues. As 125 \nmentioned above, TET1 and TET2 are highly expressed in mESCs, whereas TET3 expression is induced 126 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nin EBs and upon differentiation to neuronal lineages.  Similarly, although the DBHS protein PSPC1 127 \nrobustly associates with TET complexes in all cells tested, PSPC1 can form either PSPC1 -PSPC1 128 \nhomodimers or PSPC1-NONO and PSPC1-SFPQ heterodimers, depending on the relative abundance of 129 \neach protein (Knott et al. 2016, 2022). Of note, estimation of absolute protein abundance in wildtype 130 \nmESCs show s that TET proteins and PROSER1 are present at similar copy numbers , whereas the 131 \nabundance of OGT, PSPC1, and NONO are orders of magnitude higher (Fig. 2E). This implies that excess 132 \nOGT, PSPC1, and NONO are likely to be involved in processes independent of TET proteins and 133 \nPROSER1. In contrast, most of the cellular pool of PROSER1 may be engaged within TOPD complexes. 134 \nInterestingly, PROSER1 IP-MS did not result in enrichment of SIN3A or HDAC1/2  (Fig. 1A, 1B and sup. 135 \ntable S3), suggesting that TET interactions with the SIN3A-HDAC deacetylase complex are independent 136 \nof PROSER1. We also failed to detect interactions with UTX or members of COMPASS, and profiling of 137 \nhistone modifications by quantitative mass spectrometry revealed little or no global changes in H3K4 138 \nmethylation in two independent PROSER1 knockout mESC lines (Fig. S1G).  139 \nInitial biochemical cell fractionation of mESCs demonstrated that PROSER1 is predominantly a 140 \nchromatin-associated protein (Fig. S2 B). We therefore performed PROSER1 chromatin 141 \nimmunoprecipitation and sequencing (ChIP -seq) using our anti -murine PROSER1-N antibody to gain 142 \nfurther insights into its function in chromatin. To ensure specificity of enriched peaks, ChIP -seq was 143 \ncarried out simultaneously on wildtype and PROSER1 KO  mESCs. Analysis of biological replicate 144 \nexperiments revealed 1712 high-confidence PROSER1 binding sites (Fig. S2C). Upon intersection with 145 \npublicly available ChIP-seq datasets in mESCs, we found that a large majority (>95%) are co-occupied 146 \nby TET1, TET2, OGT , PSPC1 or combinations of these (Fig. 2F  and S2D). We next asked if PROSER1 147 \ngenome co -localisation with each of the complex components is enriched compared to matched 148 \ncontrol regions. We observed significant enrichment (P<0.0001, Fisher’s exact test) for TET1, TET2, 149 \nOGT, and PSPC1 co-localisation (Fig. 2G), whereas no enrichment was observed at CTCF binding sites 150 \nor gene bodies (Fig. S2E , F). Consistent with a role in gene regulation, PROSER1 high- confidence 151 \nbinding sites are associated with both active (H3K27ac and P300) and repressive (H3K27me3 and 152 \nSUZ12) chromatin domains (Fig. S2G) and overlap regulatory genomic regions such as promoters, CGIs, 153 \nand active enhancers which are known to be occupied by TET1 and TET2 (Fig. 2H) (Williams et al. 2011; 154 \nRasmussen et al. 2019).  155 \n   156 \nPROSER1 loss disrupts TOPD complexes and alters TET2 genome-wide chromatin binding  157 \nTo understand how loss of PROSER1 affects the stability of TOPD complexes, we immunoprecipitated 158 \nTET2 in wildtype and PROSER1 knockout mESC cell lines and analysed eluates by western blotting and 159 \nlabel-free mass spectrometry (Fig. 3A, S3A). While PROSER1, OGT and PSPC1, were present in TET2 160 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\neluates from wildtype cells, loss of PROSER1 reduced the relative recovery of OGT and abolished TET2-161 \nPSPC1 interactions altogether. Furthermore, follow-up analysis of biological triplicate IP eluates using 162 \nquantitative TMT-labeling demonstrated that TET2-OGT interactions were reduced by ~60% upon loss 163 \nof PROSER1, while quantitative recovery of SIN3A was not affected (Fig. 3B). Of note, we found that 164 \nthe level of TET2 O-GlcNAcylation was unchanged in PROSER1 knockout cells, suggesting that enzyme-165 \nsubstrate interactions between TET2 and OGT are preserved in the absence of PROSER1 (Fig. 3A). 166 \nTo investigate the association between PROSER1, TOPD recruitment to chromatin, and the 167 \nactivation status of PROSER1 -bound regions, we then performed ChIP -seq for TET2 as well as the 168 \nhistone marks H3K4me1 and H3K27ac in wildtype and PROSER1 knockout mESCs (Fig. S3B). Differential 169 \nenrichment analysis within high- confidence PROSER1 binding sites revealed that loss of PROSER1 170 \ncorrelates with reduced TET2 chromatin occupancy, while enrichment of H3K4me1 or H3K27ac does 171 \nnot change in response to loss of PROSER1 within the same regions (Fig. 3C, S3C , D, E). We observed 172 \nthat TET2 binding loss is not limited to PROSER1 binding sites within specific regulatory domains but 173 \nrather is seen across active enhancers, promoters and CpG islands (CGIs) (Fig. 3D). In contrast to the 174 \nreduction of TET2 binding at PROSER1-bound genomic regions, we also observed a significant number 175 \nof sites with increased TET2 occupancy upon knockout of PROSER1 (Fig. 3 E, F). These regions do not 176 \nshow evidence of PROSER1 binding in wildtype cells  (Fig. 3E, and S3E), or skewing towards increased 177 \nassociation with specific genomic regions compared to TET2 binding sites in wildtype cells (Fig. 3G). 178 \nHowever, these regions were linked to increases in H3K27ac upon PROSER1 knockout (Fig. 3E, 3H, S3F), 179 \npossibly via interactions of TET2 with the histone acetyltransferase P300, as reported previously (Zhang 180 \net al. 2017) . Of note, we did not  observe global differences in P300 -directed histone acetylation via 181 \nhistone mass spectrometry, nor local differences at matched control regions which were not bound by 182 \nTET2, suggesting that increased H3K27ac deposition is largely restricted to sites associated with 183 \nincreased TET2 binding (Fig. 3H, 3I, and S3G). Collectively, our results demonstrate that PROSER1 is 184 \nrequired for the stability of TOPD protein complexes, and that loss of PROSER1 alters the recruitment 185 \nof TET2 to chromatin.   186 \n   187 \nPROSER1 knockout unleashes TET catalytic activity and causes widespread DNA demethylation and 188 \nde-silencing of endogenous retroviruses  189 \nTo determine the effect of PROSER1 loss on DNA methylation, we harvested genomic DNA from two 190 \nindependent PROSER1 knockout mESC lines and quantified global levels of 5hmdC and 5mdC by mass 191 \nspectrometry. We observed a decrease in global 5m dC levels, as well as  slightly elevated levels of 192 \ngenomic 5hmdC - the major product of TET catalytic activity (Fig. 4A). To investigate which regions are 193 \naffected by increased TET activity,  we generated base- resolution DNA methylation profiles using 194 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nEnzymatic Methyl-Sequencing (EM-seq) in wildtype ESCs (WT), PROSER1 knockout (PROSER1 KO), and 195 \nPROSER1 knockout cells that were engineered to re-express full-length FLAG -tagged P ROSER1 196 \n(KO+Rescue) (Fig. 4B). Mapping of EM -seq reads to the mouse genome allowed quantification of 197 \ncytosine modification states at ~17.5 million CpG sites with at least 10X coverage in all three genotypes. 198 \nConsistent with our mass spectrometry results, we observed a decrease in DNA methylation in 10 kb 199 \nwindows across the entire genome in P ROSER1 KO cells, while reintroduction of P ROSER1 restored 200 \nmethylation to wildtype levels (Fig. 4C). This widespread DNA hypomethylation was also observed 201 \nwhen comparing average DNA methylation levels in diverse genomic regions including 202 \nheterochromatin, gene bodies, active enhancers and non-CGI promoters (Fig. 4D), though it was noted 203 \nthat regions generally depleted of DNA methylation, such as CGIs and bivalent promoters, were 204 \nunchanged (Fig. S4A). We furthermore found significant DNA hypomethylation at sites associated with 205 \nincreased H3K27ac deposition  identified previously (Fig. 4E and S3B). Importantly, expression and 206 \nprotein copy numbers of the major DNA methylation effectors (DNMT1/UHRF1, DNMT3A/B, TET1 and 207 \nTET2) were largely unchanged (Fig. 4F, S4B). This implies that DNA methylation changes are a direct 208 \nconsequence of altered TET activity in P ROSER1 knockout cells rather than a result of a general 209 \ndisruption of DNA methylation maintenance machinery.   210 \n  Dual pharmacological inhibition of DNA methylation enzymes and histone deacetylases causes 211 \nDNA hypomethylation and increased histone acetylation - reminiscent of changes observed upon 212 \nPROSER1 knockout – and synergizes to cause de-silencing of transposable elements (TEs) (Brocks et al. 213 \n2017; Daskalakis et al. 2018; Goyal et al. 2023; Cusack et al. 2020) . We therefore used a combination 214 \nof RNA-seq and TEtranscripts  - an analysis pipeline designed to handle reads that map to multiple 215 \nlocations in the genome - to assign multi -mapping reads to specific TE families and analy ze their 216 \nactivity. Loss of PROSER1 led to an increase in transcription of multiple families of long terminal repeat 217 \n(LTR)-containing endogenous retroviral (ERV) elements, such as ERVK, ERVL, and ERVL-MaLR, whereas 218 \nexpression of the non-LTR L1Md retrotransposons was mildly reduced (Fig. 4G). To understand if this 219 \nderegulation correlate d with loss of PROSER1, we assessed transcript levels of the differentially 220 \nexpressed TE families upon re- expression of PROSER1. Consistent with the observed  restoration of 221 \nDNA methylation levels described above, expression of differentially expressed TE families (Fig. 4H) – 222 \nas well as expression of individual TE elements identified solely based on uniquely mapped reads (Fig. 223 \n4I and S4C) – were restored to near -wildtype levels upon reintroduction of  full-length P ROSER1. 224 \nCollectively, our findings demonstrate that PROSER1 safeguards against genome- wide DNA 225 \ndemethylation and aberrant activation of endogenous retroviruses.     226 \n   227 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nDefective recruitment of TET2 to developmental genes upon PROSER1 loss leads to their subsequent 228 \ndysregulation during neuronal differentiation 229 \nTo determine the direct effect of altered TET2 chromatin binding , we quantified DNA methylation 230 \nchanges in the high -confidence TET2 differentially bound sites  (p- adj<0.05, abs(fold change) ≥2) 231 \nidentified in PROSER1 knockout cells (Fig. 3E). In contrast to regions with gained TET2 binding - which 232 \nmirrored the genome wide DNA hypomethylation  - sites with reduced TET2 binding were instead 233 \ncorrelated with increased levels of DNA methylation (Fig. 5A). Further analysis identified that nearly a 234 \nthird of sites with reduced TET2 binding exhibited a significant rise  in average DNA methylation (p-235 \nadj<0.05, minimum 3 CpG per site ) (Fig. 5B). To investigate potential effects on gene expression, we 236 \nidentified enriched gene ontology terms in the subset of 866 genes whose regulatory domains (defined 237 \nin GREAT as “Basal plus extension” (McLean et al. 2010) ) overlapped with sites with reduced TET2 238 \nbinding. This analysis revealed significant associations with early development processes, such as 239 \nnervous and skeletal system development, and was linked to mouse knockout phenotypes exhibiting 240 \ncraniofacial abnormalities including eye defects (Fig. 5C). In contrast, genes whose regulatory domains 241 \noverlapped with sites with increased TET2 binding were largely linked to phenotypes associated with 242 \nabnormal hematopoietic differentiation (Fig. S5A).   243 \nTo directly assess the effect of P ROSER1 knockout during mESC differentiation, we analysed 244 \ngene expression in self -renewing mESC lines and in cells differentiated for 2 days towards neuronal 245 \nlineages (N2B27 monolayer differentiation system). Initial inspection of RNA-seq results revealed that 246 \nall cell lines successfully exited pluripotency and upregulated markers of early neuronal differentiation 247 \n(Fig. S5B). Consistent with this, principal c omponent analysis (PCA) revealed differentiation state as 248 \nthe main factor driving variation in the samples (PC1: 50.8% and PC2: 42.3%). In addition, we identified 249 \na minor component (PC3: 4.1%) that correlated with P ROSER1 expression, and clearly separated 250 \nsamples cultured for 2 days in N2B27 medium (Fig. 5 D). We therefore examined if the reduced TET2 251 \nchromatin binding we observed in PROSER1 knockout cells could be linked to gene expression changes 252 \nat this developmental stage. To do this, we focused on a subset of 60 genes that i) had a regulatory 253 \ndomain overlapping sites of reduced TET2 binding in mESCs, and ii) were differentially expressed (p-254 \nadj<0.05) between wildtype and PROSER1 knockout cells upon 2 days of neuronal differentiation (Fig. 255 \n5E and sup. table S7). Contingency analysis revealed significant enrichment of this subset of genes 256 \n(both increased and decreased in knockout) compared to the subset of differentially expressed genes 257 \nnot associated with reduced TET2 binding sites (7.43% vs 3.91% of total, p-value<0.0001, Fisher’s exact 258 \ntest). Hierarchical clustering identified three clusters (C1-C3) with different expression patterns broadly 259 \nclassified as up -, down - o r  m i x e d  r e g u l a ti o n  i n  PROSER1 knockout cells, respectively (Fig. 5E) . The 260 \nobserved increases and decreases in transcript levels upon PROSER1 depletion suggest that TOPD 261 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\ncomplexes possess both activating and repressive regulatory capacities, the specific outcome of which 262 \nis likely influenced by genomic location and developmental stage. Importantly, comparison of Log2 fold 263 \nchanges in PROSER1 KO vs WT , and KO+Rescue vs WT showed that re-introduction of PROSER1 restored 264 \nexpression of many of these genes – particularly in C1 – to near-wildtype levels (Fig. 5F). Genes co-265 \nbound by PROSER1/TET2 and whose expression correlated with P ROSER1 expression included 266 \nimportant developmental regulators such as homeobox genes (e.g. Pitx2, Barx1, and Pax6), epigenetic 267 \nregulators (e.g. Cbx4, Cbx8, and Prdm8), and transcription factors (e.g. Tbx15 and Mef2c) implying that 268 \nPROSER1 plays a regulatory role in their function during early embryonic development  (Fig. S5C, D). 269 \nWe furthermore noted significant deregulation of Eyes absent homolog 4 ( Eya4) – a PROSER1/TET2 270 \nco-bound target gene – that is crucial for eye, heart and sensorineural development  (Fig. S5C , D ) 271 \n(Tadjuidje and Hegde 2013). Collectively, deregulation of these genes may underlie some, if not all, of 272 \nthe neurodevelopmental defects observed upon loss of PROSER1 in mice and humans.  273 \nIn summary, our results are consistent with a dual role for PROSER1 in directing DNA 274 \ndemethylation in early development  (Fig. 5G). We show that PROSER1 is a pan -TET interactor that 275 \npromotes the assembly of TOPD protein complexes and facilitate s their recruitment to chromatin in 276 \nthe proximity of important developmental genes. The recruitment of TET proteins – particularly TET2 277 \n– at PROSER1-bound sites maintains a lowly methylated state at regulatory regions and appropriate 278 \nexpression of adjacent genes during differentiation. Our findings also indicate  that TOPD complexes 279 \nsequester TET proteins away from other regions of the genome. When PROSER1 is depleted , TET 280 \nproteins bind to additional sites, resulting in widespread DNA demethylation , and therefore  global 281 \nDNA hypomethylation. Interestingly, the regions with increased TET2 binding also displayed increased 282 \nlevels of H3K27 acetylation. This suggests that P300, a histone acetyltransferase previously shown to 283 \ninteract with TET2  (Zhang et al. 2017) , might be recruited alongside TET2 to these same sites. The 284 \ncombined effects of reduced DNA methylation and increased chromatin openness (caused by P300 285 \nactivity) cooperate to perturb the silencing of endogenous retroviral elements, potentially disrupting 286 \nthe expression of adjacent genes during differentiation.  287 \nThe exact m echanism b y  w h i c h  T O P D  c o m p l e x e s  c o n t r o l  T E T  a c ti v i t y  a c r o s s  t h e  g e n o m e  288 \nremains unclear. One hypothesis is that certain PROSER1 binding sites act as \"sinks,\" attracting TOPD 289 \ncomplexes and TET proteins to specific genomic sites and preventing widespread, uncontrolled DNA 290 \ndemethylation by TET enzymes. Another interesting possibility involves the RNA -binding TOPD 291 \ncomponent PSPC1, which we have found to interact with TET2 in a PROSER1-dependent manner (Fig. 292 \n3A and S3A). DBHS proteins including PSPC1 can form higher-order oligomers (Fox et al. 2018; Knott 293 \net al. 2016, 2022),  potentially functioning as RNA- or DNA-tethered condensates that sequester TOPD 294 \ncomplexes, further regulating TET activity. Consistent with this, biochemical fractionation experiments 295 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nin mESCs revealed that the ability of TET2 to fractionate with chromatin is largely dependent on both 296 \nPSPC1 and its RNA binding capacity  (Guallar et al. 2018) . Additional work will be needed to address 297 \nthese possibilities in further detail.  298 \nOur findings align with prior research. Recent work revealed that introduction of a missense 299 \nmutation into Tet1 in mESCs leads to a partial disruption of TET1-OGT interaction. This results in global 300 \nDNA hypomethylation, consistent with the mutated TET1 protein detaching from TOPD complexes to 301 \ntrigger widespread DNA demethylation (Hrit et al. 2018). Similarly, a recent study observed global DNA 302 \ndemethylation and TE de-repression after acute deletion of OGT in mESCs (Sepulveda et al. 2024). This 303 \nstrongly suggests that OGT loss disrupts TOPD complex stability, leading to the release of TET proteins 304 \nand subsequent genome -wide DNA demethylation.  Interestingly, the absence of TET enzymes 305 \nthemselves has been linked to widespread DNA demethylation, particularly in heterochromatin  306 \n(López-Moyado et al. 2019) . This phenomenon was proposed to stem from disruption of a shared 307 \nprotein complex important  for both TET and DNMT enzyme recruitment.  When TET enzymes are 308 \nremoved, this complex is disrupted, leading to the redistribution of DNMT enzymes across the genome 309 \n(López-Moyado et al. 2019) . Thus, in addition to the role of PROSER1 in restraining TET activity, it is 310 \ninteresting to speculate whether TOPD complexes may have additional roles – directly or indirectly - in 311 \nregulating DNMT activity.  312 \nPair-wise TET interactions with specific partner proteins like OGT, PSPC1, and NONO have 313 \npreviously been identified (Chen et al. 2013; Vella et al. 2013; Deplus et al. 2013; Guallar et al. 2018; 314 \nHuang et al. 2022; Li et al. 2020) . However, our results imply that these interactions do not occur in 315 \nisolation but can combine to form larger multimeric TOPD complexes that have functional roles in 316 \nchromatin. Interestingly, mutations in several components of TOPD complexes beyond PROSER1 have 317 \nbeen linked to neurodevelopmental disorders. These include X -linked variants of OGT (causing 318 \nCongenital Disorder of Glycosylation (OGT-CDG))(Pravata et al. 2020; Authier et al. 2024)  and NONO 319 \n(causing NONO-associated syndromic disorder)(Mircsof et al. 2015; Reinstein et al. 2016; Roessler et 320 \nal. 2023) as well as biallelic loss of TET3 (causing Beck-Fahrner syndrome)(Beck et al. 2020; Seyama et 321 \nal. 2022), all of which display syndromic developmental delay, intellectual disability and craniofacial 322 \ndysmorphisms similar to features observed upon inactivation of PROSER1. While mutations in TOPD 323 \ncomponents are likely to have additional pleiotropic effects, some common features across these 324 \ndisorders may stem from shared disruption of TOPD complexes during development.  325 \nChromatinopathies represent an expanding category of congenital developmental disorders 326 \narising from disruptions in  chromatin function and dysregulation of the epigenome (Nava and 327 \nArboleda 2024). Mutations within genes encoding critical epigenome regulators, encompassing both 328 \ncore components and accessory proteins such as PROSER1, contribute  to this growing list of 329 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\npathologies. In this study, we show that PROSER1 plays a central role in the assembly of multi-protein 330 \nchromatin-associated TET complexes that shape the DNA methylome and support gene expression. 331 \nFurthermore, mice  lacking PROSER1 mirror the developmental defects seen in humans with 332 \nhomozygous PROSER1 loss-of-function mutations . We therefore propose that developmental  333 \nsyndromes caused by PROSER1 mutations should be designated as a novel form of chromatinopathy  334 \nand that f uture investigations into TOPD -related developmental syndromes should leverage the 335 \ngrowing understanding of TET enzyme function in development and disease.  Our development of a 336 \nPROSER1 knockout mouse model serves both as a system to gain insight into the underlying 337 \nmechanisms, and a preclinical model to explore the potential for therapeutic intervention in PROSER1-338 \nrelated developmental syndromes. 339 \n 340 \nMATERIALS AND METHODS 341 \nSee supplementary methods for further information. 342 \n 343 \nDATA AVAILIBILITY 344 \nRaw and processed data sets are available for download at the Gene Expression Omnibus (GEO) 345 \ndatabase under the accession number GSE273517.  346 \n 347 \nCOMPETING INTEREST STATEMENT 348 \nThe authors declare no competing interests 349 \n 350 \nACKNOWLEDGEMENTS 351 \nThe authors thank members of the Rasmussen lab, G. Saredi, and T. Owen -Hughes for advice and 352 \ndiscussion. We thank A. Rennie and R. Clarke in the Flow Cytometry unit, C. Gillian in the Biological 353 \nResource Unit, A. Atrih and C. Rogers in Fingerprints proteomics facility at University of Dundee as well 354 \nas A. Tavares and C. Corral in the imaging facility at the University of Edinburgh for technical assistance. 355 \nWork in the Rasmussen lab was funded by a Cancer Research UK fellowship (C66224/A27092) and 356 \nthrough support by University of Dundee. X.L. was funded through a personal scholarship provided by 357 \nthe China Scholarship Council (CSC). Work in the Hajkova laboratory is supported by MRC funding 815 358 \n(MC_US_A652_5PY70) and an ERC grant (ERC- CoG-648879–dynamicmodifications). The Sidoli lab 359 \ngratefully acknowledges, for funding , the Hevolution Foundation (AFAR), the Einstein -Mount Sinai 360 \nDiabetes center, and the NIH Office of the Director (S10OD030286). The authors thank the dedicated 361 \nteam behind the European Galaxy server (UseGalaxy.eu), supported by the German Federal Ministry 362 \nof Education and Research grant 031L0101C and de.NBI-epi.  363 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 364 \nAUTHOR CONTRIBUTIONS 365 \nA.F and K.D.R. conceived the study. A.F., E.V.K., X.L., and K.D.R. carried out experiments. A.Z., and D.O.C. 366 \ngenerated the PROSER1 knockout mouse model. Z.H. and P .H. conducted 5h mdC/5mdC mass 367 \nspectrometry experiments and analysis. S.S. and S.S. conducted histone mod mass spectrometry 368 \nexperiments and analysis. A.J.B. analyzed mass spectrometry data. 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Mol Cell 65: 323–335. 511 \nhttp://linkinghub.elsevier.com/retrieve/pii/S1097276516308516. 512 \nZhu F, Zhu Q, Ye D, Zhang Q, Yang Y, Guo X, Liu Z, Jiapaer Z, Wan X, Wang G, et al. 2018. Sin3a-Tet1 interaction activates 513 \ngene transcription and is required for embryonic stem cell pluripotency. Nucleic Acids Res 46: 6026–6040. 514 \n 515 \n 516 \n  517 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 518 \nFigure 1 Loss of PROSER1 increases preweaning lethality and is associated with developmental 519 \ndisabilities and craniofacial abnormalities [A] Histogram showing distribution of genotypes at 520 \nweaning for 476 pups born from heterozygous Proser1 +/- breeder pairs. The graph shows expected 521 \nMendelian numbers (Expected) compared to the actual distribution of genotypes (Observed).  The 522 \nnumber of homozygous PROSER1 knockout pups at weaning are demonstrably lower than anticipated, 523 \nwith less than 50% being recovered. Statistical significance for contingency table was measured by 524 \nF i s h e r ’ s  e x a c t  t e s t  ( p < 0 . 0 0 0 1 ) .  [ B ]  B o x  p l o t  s h o w i n g  b o d y  w e i g h t  i n  P R O S E R 1  k n o c k o u t  ( K O )  a n d  525 \nwildtype (WT) littermates upon reaching adulthood (~8 weeks of age). Comparisons are made for 526 \nmales and females separately ( n=8-10). Statistical significance was measured using two -way ANOVA 527 \nwith multiple comparisons (** p<0.01, *** p<0.001). [C] Bar chart showing percentages of gross 528 \nabnormalities observed in adolescent and adult PROSER1 knockout animals (n=50). Eye defects include 529 \nmicrophthalmia, anophthalmia, corneal ulcers, and cataracts. Other craniofacial defects include 530 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nintermittent head tilt and distorted ear (indicative of otitis media) as well as one case of suspected 531 \nhydrocephalus. [D] Image of a representative PROSER1 KO animal with microphthalmia. The affected 532 \neye is indicated by an arrow. [E] Dot and line graph representing volumetric analysis of microcomputed 533 \ntomography (microCT) scans of PROSER1 KO and wildtype littermate skulls (n=7). Statistical significance 534 \nwas measured by paired two-tailed t test (* p<0.05) [F] Same as E but representing average CT density 535 \n(Hounsfield Units) as an indication of bone mineral density. ns indicates not significant. [G] Lateral 536 \nviews of representative microCT scans of a 13-week-old male (above) and 14-week-old female (below) 537 \nPROSER1 KO animal as well as wildtype littermates. The images are rendered as maximum intensity 538 \nprojections (MIPs) from an equal sized volume to enable direct visual comparison between specimens. 539 \nArrows indicate malformations of maxilla and frontal bones as well as rounded head shape. Scale bar 540 \nindicates skull length of wildtype littermates. 541 \n  542 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 543 \nFigure 2 PROSER1 is a pan-TET interactor and participates in chromatin-associated TOPD complexes 544 \n[A] Volcano plot showing protein hits from α-PROSER1-C immunoprecipitation and mass spectrometry 545 \n(IP-MS) in WT mESCs (n=3 biological replicates). Protein enrichment is compared to parallel IP -MS in 546 \nPROSER1 KO mESCs. Dotted lines indicate 2 -fold change and p- value 0.05. Proteins of interest are 547 \nhighlighted and identified. See sup. table S3 for full list of enriched proteins . [B] As in A, but carried 548 \nout in cells differentiated for 2 days to mouse embryoid bodies (mEBs) where TET3 expression is high 549 \ncompared to mESCs. See sup. table S4 for full list of enriched proteins. [C] As in A, but carried out using 550 \nα-TET2-N antibody and protein enrichment compared to parallel IP -MS in TET2 KO mESCs. See sup. 551 \ntable S6 for full list of enriched proteins. [D] Illustration of the potential composition of TOPD 552 \ncomplexes in mESCs and/or mEBs. The DBHS protein dimer is depicted as consisting of PSPC1 and 553 \nNONO for simplicity but may be variable in vivo. [E] Copy number estimation (Wiśniewski et al. 2014) 554 \nof TET proteins and TET interactors in mESCs determined by MS (n=3 biological replicates). The dotted 555 \nline indicates the sum of TET1, TET2, and TET3 copies, error bars represent the mean ±S.D. [F] ChIP -556 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nseq tracks showing a representative region bound by PROSER1, TET2, OGT and PSPC1 in WT mESCs. 557 \nPROSER1 ChIP-seq in WT and PROSER1 KO mESCs was carried out in this study ; sources of the other 558 \ndatasets are indicated in the figure. Protein coding genes in the region are indicated below the tracks. 559 \nCGIs are indicated in gray. [G] Percentage of PROSER1-N peaks or matched controls ( matched to the 560 \nPROSER1-N peakset in number, size, and distance to DNase hypersensitivity sites in mESCs and 561 \ngenerated using Easeq (Lerdrup et al. 2016))  that overlap with TET1, TET2, PSPC1 or OGT binding. All 562 \npeaksets but PROSER1 were generated from publicly available data (Huang et al. 2022; Rasmussen et 563 \nal. 2019; Vella et al. 2013; Williams et al. 2011)  * p<0.0001, two -tailed Fisher’s exact test. [H] 564 \nPercentage of PROSER1-N, TET1 or TET2 peaks or matched controls that overlap with active enhancers 565 \nor promoters.  566 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 567 \nFigure 3 PROSER1 loss disrupts TOPD complexes and alters TET2 genome -wide chromatin binding 568 \n[A] Western blot of input and 10X concentration eluates from α-T E T 2  I P  o n  l y s a t e s  f r o m  W T  a n d  569 \nPROSER1 KO1 and 2 mESCs. The intensity of OGT in each eluate was normalized  to the intensity of 570 \nTET2 in the same eluate and the ratios in each IP provided beneath the figure . A slight difference in 571 \napparent molecular weight was observed between inputs and eluates owing to different buffer 572 \ncompositions. * indicates a non-specific reactive band. [B] Ratios of OGT:TET2 and SIN3A:TET2 in TMT-573 \nlabelled quantitative mass spectrometry analysis of TET2 immunoprecipitate in WT and PROSER1 KO 574 \nmESCs. TMT reporter intensity was quantified from 3 biological replicates. Error bars represent the 575 \nmean ±S.D. * p<0.05, unpaired two -tailed t-test with Welch’s correction. [C] Fold change in TET2 576 \nbinding based on TET2 normalized read counts within PROSER1 peaks or matched control regions upon 577 \nPROSER1 loss. PROSER1 peaks were sorted by Log2(fold change in PROSER1, KO-WT) and divided into 578 \nequal-sized quartiles (white). Controls (gray) were generated for each quartile. The effect sizes of 579 \nbinding loss compared to matched control regions were measured with Cohen’s d. * d >0.3 (small 580 \neffect), ** d >0.6 (medium effect), *** d >0.9 (large effect). ns, non -significant. [D] As C, but TET2 581 \nnormalized read counts are shown at PROSER1 sites within different genomic regions (white) as 582 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nspecified below the plot, or controls (gray) generated for the set of PROSER1 peaks within each 583 \ngenomic region. [E] Heatmaps and mean values of normalized  ChIP-seq signals for TET2, PROSER1, 584 \nH3K27ac and H3K4me3 centered at high -confidence (p-adj<0.05, abs(fold change≥2)) sites with gain 585 \nor loss of TET2 binding. Regions are ranked on p-adj values for TET2 binding between PROSER1 KO and 586 \nWT. Heatmaps were generated using DeepTools software (Ramírez et al. 2014). [F] Fold change in TET2 587 \nbinding based on TET2 normalized read counts within all significant (p-adj<0.05) differential sites with 588 \ngain or loss of TET2 binding (white) or matched control regions (gray) upon PROSER1 loss. The effect 589 \nsizes of binding loss compared to matched control regions were measured with Cohen’s d. * d >0.3 590 \n(small effect), ** d>0.6 (medium effect), *** d>0.9 (large effect). ns, non-significant. [G] Percentage of 591 \nall TET2 peaks (Rasmussen et al. 2019)  or all sites with significant ( p-adj<0.05) gain of TET2 binding 592 \nthat overlap with genomic regions as specified above the plot. [H] As F , but showing fold change in 593 \nH3K27ac. [I] Global enrichment of H3K27ac in WT and PROSER1 K O1 and 2 mESCs as measur ed by 594 \nquantitative MS. n=3 biological replicates of PROSER1 KO lines and 6 of WT. Error bars represent the 595 \nmean ±SD. Statistical significance was measured by unpaired two-tailed t-test with Welch’s correction. 596 \n 597 \n  598 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 599 \nFigure 4 PROSER1 loss unleashes TET activity and cause s widespread DNA demethylation and de -600 \nrepression of endogenous retroviruses  [A] Quantification of 5h mdC and 5 mdC by LC -MS/MS in 601 \ngenomic DNA harvested from WT, and PROSER1 KO1 and 2 mESCs. Each symbol represents a sample 602 \nharvested and processed independently ( n=5 biological replicates with each 2 technical replicates) 603 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nfrom each cell line. Statistical significance was measured by paired two -tailed t test (paired by same 604 \nday of harvest due to coordinated fluctuations in 5hmC). ns indicates not significant (* p<0.05, ** 605 \np<0.01). [B] Western blot of lysates prepared from WT , PROSER1 KO and PROSER1 KO+Rescue mESCs. 606 \nGAPDH was probed as a loading control. [C] Violin plot showing average DNA methylation in 10 kb tiles 607 \nfor CpG sites covered by minimum 10 EM -seq reads in all samples in WT, PROSER1 KO and PROSER 608 \nKO+Rescue mESCs. Lines on violin plot represent the median and quartiles. Statistical significance was 609 \nmeasured by Brown-Forsythe and Welch ANOVA test (**** p<0.0001). [D] Quantitation trend plots of 610 \nDNA methylation quantified by EM-seq in heterochromatin, gene bodies, active enhancers and non -611 \nCGI promoters for CpG sites covered by minimum 10 EM-seq reads in all samples in WT, PROSER1 KO 612 \nand PROSER1 KO+Rescue mESCs. [E] XY-scatter plot showing modification state of individual CpG sites 613 \ncovered by at least 10 EM -seq reads  that overlap  3196 regions of increased H3K27ac deposition 614 \nidentified previously (Fig. S3B) in PROSER1 KO vs WT cells. Significantly differentially methylated CpG 615 \nsites (p-adj<0.05, and minimum 25% difference) are highlighted in red for PROSER1 KO vs WT (above) 616 \nand KO+Rescue vs WT (below). [F] Volcano plot showing protein copy numbers determined by whole-617 \nproteome MS on PROSER1 KO mESCs ( n=3 biological replicates). Protein enrichment is compared to 618 \nparallel MS in WT mESCs. Dotted lines indicate 2-fold change and p-adj 0.05. Components of the DNA 619 \nmethylation machinery are highlighted and identified. [G] MA-plot showing TEtranscripts differential 620 \nexpression analysis of TEs in WT and PROSER1  KO mESCs. Each dot represents a sub -family of TE 621 \nelements and the number of individual elements included in the analysis (covered by at least one 622 \nunique read) is shown in parentheses. Red and dark red dots indicate significantly differentially 623 \nexpressed TE families at thresholds of p-value<0.05 and p-adj<0.05, respectively. [H] Symbol and line 624 \nplots comparing expression of TE families in PROSER1 KO vs WT and PROSER1 KO+Rescue vs WT. Plots 625 \ninclude TE families found to be differentially expressed (p-adj<0.05) between WT and PROSER1 KO in 626 \n[G] and split to show upregulated (above)  and downregulated (below) TE families. Statistical 627 \nsignificance was measured by paired two -tailed t test (* p<0.05, **** p<0.0001). [ I] Tracks showing 628 \nthe region surrounding ETnERV3-int_dup123 and 124 (highlighted in blue) which gain TET2 binding and 629 \nH3K27ac and become DNA hypomethylated and de-silenced in the absence of PROSER1. Data in the 630 \ntop two panels represent pooled EM-seq methylation calls. Data from WT mESCs are shown in black 631 \nand data from PROSER1 KO and PROSER1 KO+Rescue are overlaid in purple or blue, respectively. Below, 632 \ntracks represent ChIP -seq coverage in PROSER1 ChIP -seq ( n=2 biological replicates), and TET2 or 633 \nH3K27ac ChIP-seq (n=3 biological replicates). The last 6 tracks represent coverage of forward (Fw) or 634 \nreverse (Rv) transcripts identified by RNA-seq (n=2 biological replicates).  635 \n 636 \n  637 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 638 \nFigure 5 Defective recruitment of TET2 to developmental genes upon PROSER1 loss leads to their 639 \nsubsequent dysregulation during neuronal differentiation [A] Quantitation trend plots of DNA 640 \nmethylation for CpG sites covered by minimum 10 EM-seq reads in all samples in WT, PROSER1 KO and 641 \nPROSER1 KO+Rescue mESCs at high -confidence ( p-adj<0.05, Abs(fold change≥ 2)) sites with loss 642 \n(above) or gain (below) of TET2 binding upon PROSER1 KO. [B] Pie chart (left)  and XY-scatter (right) 643 \nshowing average DNA methylation within high -confidence sites with loss of TET2 binding upon 644 \nPROSER1 KO. Significantly (p-adj<0.05) hypermethylated or hypomethylated sites (min. 3 CpG per site, 645 \nmin. 10 reads per CpG) are indicated. [C] Bar charts showing enriched gene ontology (GO) terms 646 \nidentified by GREAT (McLean et al. 2010)  for genes with a regulatory domain overlapping high -647 \nconfidence sites with loss of TET2 binding upon PROSER1 KO. [D] PCA plot of RNA -seq data. PCA was 648 \ncarried out using Z-scores of the top 2500 variable genes in WT, PROSER1 KO1 and PROSER1 KO+Rescue 649 \ncells in the mESC state (circular markers) or after 2 days of differentiation towards neuronal progenitors 650 \n(square markers). [E] Heatmap showing Z-scores of genes differentially expressed at day 2 of neuronal 651 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\ndifferentiation between PROSER1 KO and WT (p-adj<0.05) with regulatory domains overlapping high-652 \nconfidence sites with loss of TET2 binding upon PROSER1 KO in mESCs. Hierarchical clustering 653 \nidentified three distinct clusters C1-C3 as indicated. Gene symbols for selected rows in the heatmap 654 \nare given on the right of the plot. See sup. table S7 for full list of differentially expressed genes  655 \nassociated with loss of TET2 binding. [F] Bar charts showing average Log 2 fold change for each 656 \nindividual cluster defined in [E] for PROSER1 KO vs WT and KO+Rescue vs WT in mESCs and after 2 days 657 \nof neuronal differentiation respectively. Statistical significance was measured by Brown-Forsythe and 658 \nWelch ANOVA test (* p<0.05, **** p<0.0001). [G] Schematic illustrating the dual roles of PROSER1 in 659 \ndirecting TET function and DNA demethylation. 660 \n 661 \n  662 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 663 \nFigure S1 [A] DNA and amino acid sequences of the region surrounding the PROSER1 gRNA binding 664 \nsite in WT and PROSER1KO mice. Green text indicates the start codon, blue the PAM sequence, purple 665 \nthe splice-site consensus sequence. The BstBI restriction site is indicated above the DNA sequence. 666 \nPROSER1KO mice contains a 7bp deletion that disrupts the reading frame and introduces a premature 667 \nstop codon . [B] Representative agarose gel showing genotyping of a wildtype (WT) , PROSER1  668 \nheterozygous (+/-), and knockout (KO) mouse. bp indicates base pairs [C] Western blot of lysates from 669 \nthe bone marrow of a WT or PROSER1KO mouse. GAPDH was probed as a loading control. [D] As in A, 670 \nbut in three clonal PROSER1 KO mESC lines. When an allele has no purple regions this indicates that 671 \nthe splice-site consensus sequence is lost. Premature stop codons are indicated with red asterisks. 672 \nBoth alleles in these three clones either had a frameshift mutation or lost the splice -site consensus 673 \nsequence. [B] Western blot of lysates from WT, TET2 KO or PROSER1 KO clonal mESCs. GAPDH was 674 \nprobed as a loading control. [C] Illustration of Mus musculus PROSER1 (not to scale), with the binding 675 \nregions of our PROSER1 -N and PROSER1 -C antibodies indicated below. The single letter amino acid 676 \ncodes and the residue numbers at which the indicated regions begin and end are given above. [D] 677 \nGlobal enrichment of H3K4me1, 2 or 3 in WT and PROSER1 KO1 and 2 mESCs as measured by 678 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nquantitative MS. Error bars represent the mean ±SD. Statistical significance was measured by unpaired 679 \ntwo-tailed t-test with Welch’s correction. 680 \n  681 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 682 \nFigure S2 [A] Volcano plot showing protein hits from α- PROSER1-C immunoprecipitation and mass 683 \nspectrometry (IP-MS) in TET2 KO mEBs (n=3 biological replicates). Protein enrichment is compared to 684 \nparallel IP-MS in PROSER1 KO mEBs. Dotted lines indicate 2-fold change and p-value 0.05. Proteins of 685 \ninterest are highlighted and identified. See sup. table S5 for full list of enriched proteins. [B]  Western 686 \nblot of fractionated WT, TET2 KO and PROSER1 KO1 and 2 mESCs. MEK1/2 and histone H3 were run as 687 \nfractionation controls for the cytoplasmic and chromatin fractions, respectively. * indicates a non -688 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nspecific reactive band. [ C] Volcano plot and frequency histogram showing changes in PROSER1 -689 \nchromatin binding identified by PROSER1-N ChIP-seq in WT and PROSER1 KO1 mESCs ( n=2 biological 690 \nreplicates). Dotted lines indicate 2-fold change and p-adj 0.05. PROSER1-chromatin binding sites where 691 \np-adj<0.05 and abs(fold change≥2) are highlighted in purple and their number given. [D] Venn diagram 692 \nshowing overlap between PROSER1, OGT, TET2, PSPC1 and TET1 peaks in mESCs. The number of 693 \noverlapping peaks in each combination of peaksets is indicated. All peaksets but PROSER1 -N peaks 694 \nwere generated from publicly available data (Huang et al. 2022; Rasmussen et al. 2019; Vella et al. 695 \n2013; Williams et al. 2011). [E] Percentage of PROSER1-N peaks or matched controls that overlap with 696 \nCTCF binding. CTCF binding sites were obtained from publicly available data (Song et al. 2022) . * 697 \np<0.0001, two-tailed Fisher’s exact test. [F] As E, but overlapping gene bodies. [G] Heatmaps and mean 698 \nvalues of normalized ChIP-seq signals for PROSER1 -N and activating ( P300 and H3K27ac) and 699 \nrepressive (H3K27me3 and SUZ12 ) chromatin features centered at 1712 high -confidence PROSER1 700 \nbinding sites in mESCs. P300  and SUZ12 enrichment were generated from (Wang et al. 2017)  and 701 \n(Højfeldt et al. 2018) respectively, and regions were ranked based on the H3K27me3 ChIP-seq signal. 702 \nHeatmaps were generated using DeepTools software (Ramírez et al. 2014). 703 \n  704 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 705 \nFigure S3 [A] Estimated protein abundance indicated by exponentially modified protein abundance 706 \nindex (emP AI) of TET2, OGT , PSPC1 and PROSER1 in TET2 IP-MS normalized to TET2 IP efficiencies in 707 \neach experiment (n= 1 biological replicate). n.d., not detected. [B] Volcano plot and frequency 708 \nhistograms showing changes in TET2 (left), H3K4me1 (center) or H3K27ac (right) enrichment on 709 \nchromatin identified by ChIP-seq in WT and PROSER1 KO1 mESCs (n= 3 biological replicates). Dotted 710 \nlines indicate 2 -fold change and p- adj 0.05. Sites where p-adj<0.05 and Abs(fold change ≥2) are 711 \nhighlighted in blue or black (for TET2 or H3K4me1 and H3K27ac, respectively) and their number is 712 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\ngiven. [C] Fold change in PROSER1 binding (left) or TET2 binding (right) based on normal ized read 713 \ncounts within PROSER1 DOWN and PROSER1 UP sites (p-adj<0.05) (white) or matched control regions 714 \n(gray) upon PROSER1 loss. The effect sizes of binding loss compared to matched control regions were 715 \nmeasured with Cohen’s d. * d>0.3 (small effect), ** d>0.6 (medium effect), *** d>0.9 (large effect). ns, 716 \nnot-significant. [D] Fold change in PROSER1 binding (above) or enrichment of H3K4me1 (center) or 717 \nH3K27ac (below) within PROSER1 peaks or matched control regions upon PROSER1 loss. PROSER1 718 \npeaks were sorted by Log 2(fold change in PROSER1, KO- WT) and divided into equal -sized quartiles 719 \n(white). Controls (gray) were generated for each quartile. The effect sizes of binding loss compared to 720 \nmatched control regions were measured with Cohen’s d. * d >0.3 (small effect), ** d >0.6 (medium 721 \neffect), *** d>0.9 (large effect). ns, non-significant. [E] Percentage of PROSER1 peaks that overlap with 722 \nhigh-confidence (p-adj<0.05, Abs(fold change≥2)) sites with gain or loss of TET2 binding. * p<0.0001, 723 \ntwo-tailed Fisher’s exact test. [F] As D, but fold change in TET2 binding ( left) or H3K27ac enrichment 724 \n(right) at quartiles of all sites with significant (p-adj<0.05) gain of TET2 binding. [G] Global enrichment 725 \no f  H 3 K 5 a c  ( l e ft ) ,  H 4 K 8 a c  ( c e n t e r )  a n d  H 3 K 1 8 a c  ( r i g h t )  i n  W T  a n d  P R O S E R 1  K O 1  a n d  2  m E S C s  a s  726 \nmeasured by quantitative MS. n=3 biological replicates of PROSER1 KO lines and 6 of WT.  Error bars 727 \nrepresent the mean ±SD. Statistical significance was measured by unpaired two -tailed t-test with 728 \nWelch’s correction. 729 \n 730 \n  731 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 732 \nFigure S4 [A] Quantitation trend plots (window size 50 bp, step 50 bp, 1 kb flanking sequence) of DNA 733 \nmethylation quantified by EM-seq in bivalent promoters and CGIs for CpG sites covered by minimum 734 \n10 EM-seq reads in all samples in WT, PROSER1 KO and PROSER1 KO+Rescue mESCs. [B] Coverage of 735 \nforward (Fw) or reverse (Rv) transcripts from RNA -seq (n=2 biological replicates) in WT, PROSER1 KO 736 \nand KO+Rescue mESCs at the regions surrounding Dnmt1 (left) and Uhrf1 (right). [C] Box plots showing 737 \nTEtranscripts differential expression analysis of TEs. Dots represent expression of individual TEs whose 738 \nexpression was quantified using only uniquely mapped RNA -seq reads. The box plots compare 739 \ndifferential expression of TEs in PROSER1 K O vs WT and PROSER1 K O+Rescue vs WT for ET nERV3-int 740 \n(left), RLTR13B2 (middle), and IAPEy-int (right) families of endogenous retroviral elements, all found to 741 \nbe significantly deregulated upon PROSER1 KO. Statistical significance was measured by unpaired two-742 \ntailed t-test with Welch’s correction (* p<0.05). 743 \n 744 \n  745 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\n 746 \nFigure S5 [A] Bar charts showing enriched gene ontology (GO) terms identified by GREA T (McLean et 747 \nal. 2010) for genes with regulatory domain overlapping high-confidence sites with gain of TET2 binding 748 \nupon PROSER1 KO.  [B] Heatmap showing the changes in expression (Z-score of normalized read counts 749 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint \n\nfrom RNA-seq) of key pluripotency markers (above), and neuronal markers (below) in WT, PROSER1 KO 750 \nand PROSER1 KO+Rescue mESCs (left) or cells subjected to monolayer differentiation in N2B27 medium 751 \n(right). Gene symbols for each row in the heatmap are given on the right of the plot. [C] As in B, but 752 \nfor selected developmental genes co-bound by PROSER1 and TET2 whose expression correlated with 753 \nPROSER1 expression. [D] Tracks showing the region surrounding the developmental genes Prdm8, 754 \nEya4, Tbx15, and Cbx4. Data in the top two panels represent pooled EM -seq methylation calls. Data 755 \nfrom WT mESCs are shown in black and data from PROSER1 KO and PROSER1 KO+Rescue are overlaid 756 \nin purple or blue, respectively. Below, tracks represent ChIP-seq coverage in PROSER1 ChIP-seq (n=2 757 \nbiological replicates), and TET2 or H3K27ac ChIP-seq (n=3 biological replicates). CGIs are indicated in 758 \ngray. 759 \n 760 \n 761 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 2, 2024. ; https://doi.org/10.1101/2024.07.31.606086doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}