Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor

Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor Melis A Aksit , Bo Yu , Bernard AJ Roelen , View ORCID Profile Barbara R. Migeon doi: https://doi.org/10.1101/2021.10.05.463256 Melis A Aksit 1 McKusick Nathans Department of Genetic Medicine, Johns Hopkins University, School of Medicine , Baltimore, MD, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bo Yu 3 Farm Animal Health, Department of Population Health Sciences, Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University , 3584CM Utrecht, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bernard AJ Roelen 4 Embryology, Anatomy and Physiology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University , 3584CM Utrecht, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Barbara R. Migeon 1 McKusick Nathans Department of Genetic Medicine, Johns Hopkins University, School of Medicine , Baltimore, MD, United States of America 2 The Department of Pediatrics’, Johns Hopkins University, School of Medicine , Baltimore, MD, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Barbara R. Migeon For correspondence: bmigeon{at}jhmi.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus ( in cis ) on each additional X chromosome initiates the transcriptional silencing of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST function on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos – well before the early blastocyst (E5) when XIST on the inactive X in females begins to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos. INTRODUCTION Only one X chromosome is active in diploid human cells, irrespective of the sex of the individual and the number of X chromosomes 1 . XIST , a non-coding RNA 2 emanating from the X inactivation center, has been shown to be a potent chromosome silencer – not only for the inactive X in all the eutherian mammals studied 3 - 6 but also for autosomal chromosomes into which it has been transfected 7 , 8 . Although another non-coding RNA, antisense to XIST , seems to have a role in protecting the mouse active X, its human counterpart, TSIX , was truncated during mammalian evolution 9 , 10 , is co-expressed with XIST from the inactive X in male and female cells 10 and has not been shown to play a role in protecting the active X in species other than rodents. Further, TSIX has not been found in many mammalian genomes. ( https://genome.ucsc.edu/cgi-bin/hgGateway.6/26/21 ) Human triploid cells (69, XXX and 69, XXY) have two active X chromosomes 11 - 17 because a second X is protected from silencing by the extra set of autosomes in triploid cells. The simplest explanation is that active X’s are chosen by repressing their XIST l oci; the key repressor is encoded by an autosome 11 , 18 - 20 . Previously, we identified two candidate autosomes 11 based on a comprehensive study of human trisomies (47,XX). We could exclude the X chromosome and all autosomes, (except 1 and 19), because unlike triploids, these 21 different trisomies had only one active X 11 , 14 . Trisomies 1 and 19 could not be studied, as they do not survive implantation, presumably because they are gene-dense. Therefore, we ascertained all available partial trisomies of chromosome 1 and 19 that survive gestation. By searching the literature to determine the trisomic regions that are tolerated in liveborns, we could exclude them from our candidate regions of chromosomes 1 and 19 11 . In 2017, we could analyze duplications of chromosome 1 and 19 in the Decipher database, which records the sex and phenotypes of chromosomal duplications, deletions and single nucleotide variants 19 . Because extra doses of repressor would not be lethal for males with only one X chromosome, yet cause the death of females with two active X chromosomes, we looked for sex differences in duplications on chromosomes 1 and 19; as controls, we performed the same search for deletions on chromosomes 1 and 19, and for duplications on all the other human autosomes in the Decipher database. What we observed was that only chromosome 19 had an extensive region (~ 8 MB) on its short arm (19p13.3–13.2), that is intolerant of interstitial or tandem duplication in females 21 , suggesting that it contains the key dose-sensitive gene(s) that induces XIST repression 19 , 21 . The sex difference in duplications on chromosome 19p was highly significant (290 genes, which code for proteins as well as epigenetic factors 19 . Many of these genes could be eliminated from consideration as they are expressed in single tissues, or are expressed only after implantation, or have known functions incompatible with XIST repression. The most relevant genes were the chromatin remodelers that were expressed in every tissue. We knew that epigenetic factors could repress XIST in trans . Of particular interest were the writers and erasers of epigenetic marks, such as the lysine demethylases, which have been implicated in Xist activation in mice 22 , 23 . If lysine demethylases could activate Xist expression, other epigenetic marks might prevent it, perhaps by histone changes leading to DNA methylation. Among the relevant 19p candidate genes are the DNA methyltransferase, DNMT1 , its co-factor UHRF1 , the satellite attachment factors SAFB1 and SAFB2 , the hetero-ribonuclear protein HNRNPM , the histone methylases, KDM4B , and KDT2B , the long non-coding RNA (lncRNA) TINCR , as well as a cluster of genes coding for zinc finger proteins ( Table 1 ). We analyzed these genes as they seemed able to repress XIS T. View this table: View inline View popup Download powerpoint Table 1: The genes analyzed in human preimplantation embryos. Previous studies showed that XIST is expressed – albeit at levels that cannot silence the chromosome – from every X in human males and females at the four to eight cell stage 24 . Silencing of X chromosomal genes occurs only when XIST is upregulated from the future inactive X, beginning at the early blastocyst stage 25 , 26 . Because the XIST locus on the future active X needs to be silenced before XIST is upregulated on the future inactive X, we ascertained the time during preimplantation development when our candidate genes are transcribed. We examined RNA transcripts collected from preimplantation human embryos, using the single-cell RNA-sequencing data present in the two published datasets 25 , 26 available to us. Analyzing their expression, we identified candidate genes with patterns that suggest they could be involved in XIST repression. We validated the human expression patterns in bovine preimplantation embryos. It has been shown that the bovine is a more appropriate model for human X-inactivation than the mouse 3 . Both human and cattle are mono-ovulatory and the timing of oocyte development and embryonic genome activation as well as the embryonic transcriptomes are more similar than they are to mouse 27 . Bovine XIST is also not repressed by its antisense gene, as TSIX has not been found in the bovine genome ( https://genome.ucsc.edu/cgi-bin/hgTracks?db=bosTau9 ). Bovine, like humans and many other mammals except rodents, have conserved this block of candidate genes over millions of years of evolution and the chromosome 19p genes are clustered on bovine chromosome 7 28 . Because of the many species differences in development 18 , we did not expect the bovine to regulate X chromosomes exactly like humans, but unlike rodents they do not imprint X inactivation, they conserve the 19p gene cluster on bovine chromosome 7 and they do not use TSIX to protect the active X. MATERIALS AND METHODS Single-cell RNA Sequencing Datasets Data from single cell RNA-sequencing (scRNA-seq) of human preimplantation embryos were taken from two publicly available datasets: Dataset 1 : Yan, et al. (GEO accession: GSE36552) 26 and Dataset 2 : Petropoulos, et al (EMBL-EBI accession: E-MTAB-3929) 25 . See Supplemental Table 1 for specimens that are included in each dataset. These two datasets available for our analysis have different content of embryos. Sexing of Human Embryos using scRNA-seq Sexing of embryos of Dataset 1 26 was conducted by Moreira de Mello, et al (2017) 29 using Y-linked expressed genes outside of the pseudoautosomal region; sexing begins at the 8-cell stage ( Supplemental Table 1 ). Sexing in Dataset 2 was conducted by Petropoulos, et al (2016) 25 (Reinius, B, personal communication), also based on the expression of the Y-linked genes outside of the pseudoautosomal region as described in Petropoulos, et al (2016) 25 . Bovine in vitro embryo production and sample collections Bovine ovaries, collected from a local slaughterhouse, were transported to the laboratory in a thermos flask. After a water rinse at 30°C, the ovaries were kept in 0.9% NaCl supplemented with penicillin/streptomycin (100 µg/mL) at 30 °C. Cumulus-oocyte complexes (COCs), aspirated from follicles with a diameter of 2-8mm, were identified using a stereomicroscope. The COCs were matured in vitro and fertilized as described previously 30 . After 23h maturation, COCs were transferred to fertilization medium and incubated with 10 6 /mL sperm cells. Female or male embryos were derived from X or Y-sorted sperm (CRV, Arnhem, the Netherlands) 3 . The moment that sperm were introduced into fertilization medium was considered day 0. After incubation with sperm for 20-22h, presumptive zygotes were freed from cumulus cells by vortexing for 3 min, and placed in synthetic oviductal fluid (SOF) in a humidified atmosphere at 5% CO 2 and 7% O 2 at 39 °C1. At day 5, cleaving embryos were transferred to fresh SOF and further cultured until day 8. For subsequent analysis, zygotes, two, four and eight cell embryos were collected at 20, 32, 38 and 56 h after the start of fertilization, respectively. Morulae and blastocysts were collected on day 5 and day 8, respectively. Embryos in pools of 20 were stored in RLT buffer (Qiagen, Valencia, CA, USA) at −80 °C until RNA extraction ( see Supplementary Experimental Data ). RESULTS Expression levels of XIST , candidate XIST repressor genes and three zygotic activation marker genes ( NANOG, POU5F1 , and SOX2 ) were evaluated in two published human single-cell RNA-seq datasets ( Table 1 ), to determine when XIST repression occurred, and which candidate genes were expressed at that time. Our candidate genes included genes from the region of chromosome 19p we previously demonstrated has a sex bias 19 . NANOG, POU5F1 , and SOX2 were studied because they are known to be upregulated immediately after transcription transitions from maternal oocyte to zygote in human pre-implantation embryos 31 . Also we studied the primate specific X-linked lncRNA, XACT , as others suggest it is the gene that protects the active human X from being silenced by its XIST locus 32 . Because 47, XXX diploid cells with three copies of XACT , have only a single active X, we do not consider XACT or any other X-linked gene to be a serious candidate for the XIST repressor (see Discussion ); however, we wished to determine if XACT was expressed at the time of XIST repression. Whenever possible, we used the sex of the embryo to determine if expression of the locus occurred in males as well as females. XIST was first expressed at extremely low levels in the 8-cell or morula stages by both male and female embryos, but it showed a female sex bias in the early blastocyst stage ( Figure 1 ). In Dataset 1 26 XIST was not expressed until the 8-cell stage ( Figure 1A ; Oocyte, Zygote, 2-cell and 4-cell average expression values: 0 RPKM). XIST expression was very low in both sexes at the 8-cell stage; it increased in the morula and was greatest at the late blastocyst stages of female embryos ( Figure 1A ; 8-cell expression: 0.48 RPKM, morula (E4) average expression: 1.01 RPKM, early blastocyst (E5) average expression: 3.31 RPKM). In Dataset 2 25 the expression of XIST was also very low at the 8-cell stage (0.09 RPKM); it increased from morula (E4) to E5-E7 blastocyst stages ( Figure 1A , average expression 2.91, 4.25, 11.38 and 9.09 RPKM, in E4, E5, E6 and E7, respectively). It is clear from Dataset 2 that female embryos are the ones that express XIST in the early (E5) blastocyst (average female XIST expression: 6.61 RPKM; average male XIST expression: 0.67 RPKM), confirming that this is the time when XIST begins to be up-regulated in females – eventually silencing the future inactive X chromosomes. Download figure Open in new tab Figure 1: Box plots of gene expression (RPKM) of XIST, NANOG, POU5F1 and SOX2 determined by scRNA-seq in preimplantation human embryos. Embryos are grouped by sex and dataset (Pink: Females from dataset 1, Light blue: Males from dataset 1, No fill: undetermined sex from dataset 1, Red: Females from dataset 2, Blue: Males from dataset 2). The horizontal line in the middle indicates median value, and the boxes show the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1 . 5 * IQR from the hinge (IQR: inter-quartile range; distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1 . 5 * IQR of the hinge. Data beyond the end of the whiskers are not plotted. Significant differences between females and males are indicated by * (p < 0 . 05), ** (p < 0 . 01) and *** (p < 0 . 005) . Figure 1 also shows the expression of NANOG, POUF51 and SOX2 , that are critical for maintaining pluripotency and are expressed at the time when tissues begin to differentiate 33 , 34 .. The first two genes were not detectable until the human eight-cell – morula stages, reaching a maximum in the early blastocyst (E5). SOX2 was transcribed at the eight-cell stage and is most abundant in the morula. Females expressed NANOG at higher levels at the E5-E7 blastocyst stages. Although the expression values vary between the two datasets due to technical variance associated with the sequencing technology and between sexes due to individual variation, whether or not the gene is expressed at the various stages is consistent. Because 47, XXX females with three copies of the X-linked lncRNA, XACT , have two inactive X’s and only one active X, we had previously eliminated XACT and other X-linked genes as candidate XIST repressors. And in our analysis, we did not find that XACT was expressed in Dataset 1 ( Table 1 ). Petropolis et al 25 (Dataset 2) reported its expression at very low levels (<3 RPKM) in morulae (E4) and E5-E7 blastocysts, which is below our cut-off for being expressed (5 RPKM) ( Table 1 (see Figure S5 in Petropoulos et al 25 ). XACT may function mainly in maternal oocytes where it has been observed to be expressed from both X chromosomes in human primordial germ cells, but not in ovarian somatic cells 35 ; it has been proposed to play a role in imprinting maternal genes in primordial germ cells 35 . Our interpretation that XACT has no role in protecting the active X is supported by recent studies of human embryonic stem cells that indicate that the deletion of XACT has no effect on X inactivation, but does perturb neuronal development in those cells 36 . The evidence, including this paper, is now definitive enough to eliminate XACT as a candidate XIST repressor. Candidate genes that could be eliminated from consideration because they are not expressed during pre-implantation Among genes that were not transcribed in the preimplantation human embryo are KDM4B, PTPRS, ZNRF4, ZNF414, RFX2 and PRMT4 , and so these genes can be eliminated from our list of candidates ( Table 1 ; Supplemental Figure 1 ). SIRT6 and TINCR , which are not expressed prior to XIST upregulation, are also unlikely to be key XIST repressors ( Supplemental Figure 1 ). Single-cell RNA-sequencing of human embryos shows that DNMT1 and UHRF1 are the candidate genes most highly transcribed prior to XIST upregulation Of the genes evaluated ( Table 1 ), the DNA methylase, DNMT1 and its co-factor, UHRF1 , were expressed the earliest and at the highest levels – paradoxically, at a time when DNA demethylation of the zygotic genome is prominent 37 . In Dataset 1 , at the oocyte-4 cell stages, DNMT1 was highly transcribed (averaging 1796.8, 2460.2, 4260.0, and 2440.1 RPKM in oocyte, zygote, 2-cell and 4-cell stages, respectively) when XIST was not expressed. In contrast, the levels of DNMT1 transcription drastically decreased when XIST is upregulated (averaging 469.6, 301.3, and 4.2 RPKM, in the 8-cell, morula and late blastocyst stages, respectively), which was significantly different from the 4-cell stage (t-test p-values: 3.4e-13, 1.6e-13 and 1.5e-10 for 8-cell, morula and late blastocyst stages, respectively). Dataset 2 showed the same trends; DNMT1 was highly expressed at the 8-cell stage (mean 1005.6 RPKM), whereas its expression was much less at the morula-blastocyst stages ( Figure 2C ). Download figure Open in new tab Figure 2: Box plots of gene expression (RPKM) for UHRF1, SAFB2, DNMT1, SAFB, HNRNPM, UBL5, SMARCA4 and ZNF823 , determined by scRNA-seq in preimplantation human embryos. Significant differences between females and males are indicated by * (p < 0 . 05), ** (p < 0 . 01) and *** (p < 0 . 005) . UHRF1 , coding for the DNMT1 co-factor had a similar expression pattern. For Dataset 1 the average expression levels were 759.9, 560.1, 685.6 and 487.1 RPKM in the oocyte, zygote, 2-cell and 4-cell stages, respectively, significantly decreasing to 35.4, 3.1, and 18.7 RPKM in the 8-cell, morulae and late blastocyst stages, respectively (t-test p-values in comparison to the 4-cell stage: 6.3e-8, 8.7e-8, 1.1e-7, respectively). The results were similar for Dataset 2 ( Figure 2A ). Other candidate genes transcribed prior to XIST upregulation Albeit at a lower level than DNMT1 and UHRF1 , the genes for the satellite attachment factors SAFB and SAFB2 were expressed prior to XIST upregulation ( Figures 2 ); therefore, they remain candidates for the key XIST repressor. In addition, other genes in the region that encode nuclear proteins, HNRNPM and URL5 were also expressed prior to XIST up-regulation ( Figure 2 ). On the other hand, SMARCA4 , which was transcribed prior to XIST upregulation ( Figure 2 ) encodes a catalytic subunit of SWI/SNF complexes that remodels chromatin, making it more accessible to transcriptional activation – hence despite its transcription pattern, SMARCA4 is less likely to be the XIST repressor. Though several zinc finger proteins were not transcribed in the early embryo, ZNF823 was expressed during the four – eight cell stage ( Figure 2 ). Bovine embryos show patterns of gene expression similar to humans Because the patterns of XIST expression and silencing of the inactive X in bovine embryos are similar to those of human embryos 3 , 38 we analyzed the expression of our candidate XIST repressor genes in bovine preimplantation embryos. The availability of sexed semen enabled us to generate sex-specific embryos. To confirm the accuracy of the sex-sorted sperm, we examined the expression of DDX3Y , which is transcribed from the Y chromosome in male embryos. As expected, the expression of DDX3Y was significantly higher in male than female embryos. The low level of expression in female embryos reflects the ~ 90% accuracy of the sperm sexing ( Figure 3 ). Download figure Open in new tab Figure 3: The relative expressions of candidate XIST repressor genes in female (red bars) and male (blue bars) bovine embryos from 4-cell stage to day 8 blastocysts, as detected by quantitative RT-PCR. Embryos were derived by fertilization with sex-sorted sperm. Relative expression from male blastocysts set at 1. Significant differences between females and males are indicated by * (p < 0 . 05), ** (p < 0 . 01) and *** (p < 0 . 005). Significant differences among embryos with same gender are indicated by different letters with the same color (p < 0 . 05). Error bars indicate standard deviations of three independent biological replicates . Focusing on the expression of XIST, DNMT1 and UHRF1 , we found patterns similar to our human data. In unsexed bovine embryos, XIST expression has been detected at very low levels from the oocyte stage onwards, representing maternal transcripts 3 . The zygotic expression of XIST begins at the morula stage in both sexes and increases significantly at blastocyst stages only in female embryos ( Figure 3A ). We detected the expression of DNMT1 and UHRF1 from the zygote stage onwards, at similar levels in male and female embryos ( Figure 3D ). Like in human embryos, UHRF1 and DNMT1 expression in the bovine embryos is downregulated after activation of the zygotic genome. Because zygotic activation occurs slightly later in bovine, downregulation of UHRF1 and DNMT1 also occurs slightly later in bovine ( Figure 3 ). The expression of bovine UHRF1 was enigmatically greater in male than female four-cell embryos ( Figure 3G ). We also examined the expression of other candidate XIST repressors, HNRNPM, SAFB, SAFB2 and UBL5 , clustered on the region of bovine chromosome 7 that is homologous to the short arm of human chromosome 19. These genes were expressed throughout early preimplantation development, particularly in the morula ( Figure 3 ). DISCUSSION XIST , the long non-coding RNA is critical for silencing the inactive X. However, XIST needs to be silenced on the active X, so that the chromosome can continue to be transcribed and the promoter of the silent XIST locus on the active X is normally methylated in postnatal cells 39 . We have previously identified a region on chromosome 19 that we propose is involved in maintaining the transcriptional activity of the active X. Our comprehensive analysis of trisomies 11 , 19 has directed us to that region on chromosome 19 and eliminated all other chromosomes, including the X, as the source of the key XIST repressor. Therefore, we hypothesized that some of the genes within the relevant region of chromosome 19, specifically those that could modulate chromatin, might be candidates for the key XIST repressor. Now, we have carried out an in-depth analysis of expression levels of some of the most likely candidate genes in this region during preimplantation development in both human and bovine embryos. Our studies reveal that some of our human candidate genes are transcribed at the 4-8 cell stage ( Figures 2 and 3 ) – the time that zygotic transcription occurs in the human embryo 31 . Bovine candidate genes are also expressed at the time of zygotic transcription, which occurs from the 8 cell-morula stages. This major transcriptional reprogramming event requires prior remodeling of the zygotic chromatin for transcriptional competence, and elimination of maternal transcripts. We propose that this is also when repression of the XIST locus on the future active X must occur; our observations suggest that it occurs with the upregulation of the pluripotency factors – the time when fetal developmental programs begin. All the genes that remain candidates are involved in the regulation of chromatin. HNRNPM, SAFB, SAFB2 , and URL5 code for proteins which regulate transcription. Products from both HNRNPM and UBL5 do not associate with protein; they only bind to RNA 40 and have been implicated in the splicing of RNA. XIST has different splice variants 2 , but their importance to its function remains to be understood. Heterogeneous nuclear ribonucleoproteins, which directly bind to nascent RNA polymerase II transcripts, play an important role in processing heterogeneous nuclear RNAs to form mature mRNAs and in regulating gene expression. Ubiquitination is at the center of these mechanisms – occurring on all types of histones and regulating most nuclear signaling pathways. The products of UHRF1 and DNMT1 place methyl groups on CPGs in the promoter region of genes, thus repressing their transcription. UHRF1 codes for an E3 ligase, which is recognized by DNMT1, and their interaction stimulates the methyltransferase activity of DNMT1 – the key step in the maintenance of methylation patterns. Although DNMT1 is a maintenance methylase, it is also known to have de-novo methylating activity in the oocyte and early embryo 41 , 42 , and it is the imprinter of parentally imprinted genes 25 . In previous studies when Dnmt1 is knocked out in male mice, Xist is expressed from the active X chromosome, indicating that DNMT1 can repress XIST 39 . The SAFB binding proteins are known to be satellite attachment factors. Both SAFB binding proteins, whose genes are in head-to-head orientation on human chromosome 19p13.2, have been shown to bind XIST as well as other non-coding RNAs 43 , but the consequences of that binding are not yet known. The SAFB proteins, UHRF1 and DNMT1 and other candidates are transcribed in both sexes, as expected, because both males and females need to repress XIST on their active X. Because zygotic activation occurs after the 4 cell stage, the earlier transcripts from UHRF1, DNMT1, HNRNPM, UBL5 and the SAFB loci in oocytes and zygotes, must be maternal in origin ( Figures 2 and 3 ). Maternal RNAs are known to imprint some embryonic genes during oogenesis, including the maternal Xist gene in mice 35 . Yet, these genes must function in the fetal genome as well, as it is the duplication of 19p in the fetal genome that interferes with development of the female fetus. At the 4-8-cell stages, clearly these transcripts must originate from the fetus ( Figures 2 and 3 ). Unfortunately, we have no means to determine from the present data if the origin of these gene transcripts at any stage is maternal or fetal or a combination of the two. Conceivably, transcription of DNMT1 and URHF1 begins in the oocyte and continues in the fetus after zygotic activation, and it is the fetal transcripts that are responsible for XIST repression. Despite being >5MB apart on all the orthologs of chromosome 19p in a large number of mammalian species, the genes for methyltransferase DNMT1 and its co-factor UHRF1 remain together on that chromosome throughout millions of years of evolution 28 . Therefore, because they are transcribed from the pre-implantation embryo at the time of zygotic transcriptional activation, they are prime candidates for repression of XIST on the active X. This is also true for XIST binders SAFB and SAFB2 . Of interest, the only other satellite attachment factor, SAFA , which is encoded by human chromosome 1, has been shown to be involved in silencing the inactive X by binding to mouse Xist 44 . Our studies of the transcription of 19p genes DNMT1, URHF1 and several other 19pgenes that could repress the XIST locus on the active X of males and females reveal similar patterns of transcription in human and bovine, for considering the species differences in time of zygote activation. These studies provide further evidence that bovine is a good model for human X inactivation. As expected, the patterns are not identical, because of species variations that occurs throughout mammalian evolution. However, they suggest that transcription of the key XIST repressor may start in the oocyte, but that it is during the four to eight cell stage and morula when zygotic transcription begins, that XIST on the future active X is repressed in both males and females. Genes coding for DNMT1 , its co-factor UHRF1 , the satellite attachment factors, SAFB and SAFB2 , the RNA binding proteins, HNRNPM and URL5 , and zinc finger protein ZNF823 , all residing on human chromosome 19p and bovine chromosome 7 remain candidates for the key XIST repressor. Our results suggest that several of our candidate genes act together to repress the XIST locus on the future active X; they also provide a list of candidate genes that can be tested for their ability to repress XIST by knocking them out at the appropriate stage in human or bovine preimplantation embryos, or other appropriate non-rodent models. We also show that XACT is not the human XIST repressor, not only because of very low or non-expression in preimplantation embryos, but because it is X-linked. For most eutherian mammals, the transcriptional silencing of XIST on the future active X provides an alternative model for X dosage compensation. X inactivation does not choose the inactive X, but chooses the X that will remain active by silencing its XIST locus. There is no need to count X chromosomes, as the future active X is chosen by silencing one XIST locus in both sexes, no matter the number of X chromosomes in the cell. In males, most often that X is their only X. Based on our studies, in human females, proximity to chromosome 19p plays a role in the choice. We know little about the nature of trans -interactions between chromosomes. Future studies should reveal how this is accomplished. Contents of Supplemental Material 2 Supplemental Tables and 2 Supplemental Figures Supplemental Methods Declaration of Interests The authors declare no competing interests. Supplementary Methods RNA extraction, cDNA synthesis and qRT-PCR Total RNA was extracted using an RNeasy Micro Kit (Qiagen, Valencia, CA, USA) and complementary DNA (cDNA) synthesis was performed 3 . Primer sets ( Eurogentec ) were designed on Primer-Blast ( http://www.ncbi.nlm.nih.gov/tools/primer-blast ) using Bos taurus nucleotide sequences (Genbank; http://www.ncbi.nlm.nih.gov/nucleotide ) as the template. To determine primer specificity and optimal annealing temperature, temperature gradients ranging from 55°C to 68°C were tested, using cDNA from 100 blastocysts. All amplification reactions were performed on three independent cDNA samples in duplicate, following the manufacturer’s protocol for the CFX detection system ( Biorad ). The reaction mixture was prepared with 10 µL iQ SYBR Green supermix ( Biorad ), 9 µL RNAse- and DNAse-free water (Invitrogen) and 1 µL cDNA with a final primer concentration of 500 nM. Reactions were started at 95 °C for 3 min, followed by 40 cycles of subsequently 95 °C for 20 sec, the primer specific annealing temperature ( supplemental Table 2 ) for 20 sec and extension at 72 °C for 20 sec. To verify the purity of the PCR products, melting curves were plotted (with temperature increments of 0.5 °C) from 65 °C to 95 °C (each step for 5 sec). To determine quantitative (q) RT-PCR amplification efficiency, standard curves for each primer pair were made by 4 fold dilutions of cDNA from 400 oocytes or 100 blastocysts. Data were normalized using GAPDH, SDHA and YWHAZ as reference genes 45 . Statistical analysis Statistical analysis of data from qPCR were carried out in Microsoft Excel and statistical differences were examined using GraphPad Prism 7 ( https://www.graphpad.com/scientific-software/prism/ ). Differences between two groups were determined by two-tailed unpaired Students□t-tests and differences between multiple groups were analyzed by one-way ANOVA, followed by a post-hoc Tukey test. Statistical significance was set at P < 0.05. For human scRNA-seq statistical tests comparing gene expression over time, two-sample t-tests with unequal variance were used. View this table: View inline View popup Download powerpoint Supplementary Table 1: Distribution of embryos and cells in various stages in data obtained by Petropoulos, et al. and Yan, et al. View this table: View inline View popup Download powerpoint Supplementary Table 2: Primers used for quantitative RT-PCR analysis Download figure Open in new tab Supplemental Figure 1: Box plots of gene expression (RPKM) determined by scRNA-seq in preimplantation human embryos for SIRT6 and TINCR. Download figure Open in new tab Supplemental Figure 2: The relative expression of XCI related genes during in vitro bovine embryo development, as determined by quantitative RT-PCR. (A) DNMT1, (B) UHRF1, (C) HNRNPM, (D) UBL5, (E) SAFB, (F) SAFB2. Embryos were generated by fertilization with non-sexed sperm. Relative expression in GV oocytes is set at 1. Significant differences between bars are indicated by different letters above bars (p < 0.05). Error bars indicate standard deviations of three independent biological replicates. Acknowledgements The authors are grateful to our Hopkins colleagues, Michael Beer, Hans Bjornsson, Haig Kazazian, Garry Cutting, Jeremy Nathans for their careful reading of the paper and their insightful suggestions. We also thank Drs. Bjorn Reinius and Frederik Lanner for their help with the sexing of embryos. References 1. ↵ Lyon , M.F. ( 1962 ). Sex chromatin and gene action in the mammalian X-chromosome . Am J Hum Genet 14 , 135 – 148 . OpenUrl PubMed Web of Science 2. ↵ Brown , C.J. , Hendrich , B.D. , Rupert , J.L. , Lafrenière , R.G. , Xing , Y. , Lawrence , J. , and Willard , H.F. ( 1992 ). The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus . Cell 71 , 527 – 542 . doi: 10.1016/0092-8674(92)90520-m . OpenUrl CrossRef PubMed Web of Science 3. ↵ Yu , B. , van Tol , H.T.A. , Stout , T.A.E. , and Roelen , B.A.J. ( 2020 ). Initiation of X Chromosome Inactivation during Bovine Embryo Development . Cells 9 . doi: 10.3390/cells9041016 . OpenUrl CrossRef 4. Lee , J.T. , and Jaenisch , R. ( 1997 ). Long-range cis effects of ectopic X-inactivation centres on a mouse autosome . Nature 386 , 275 – 279 . doi: 10.1038/386275a0 . OpenUrl CrossRef PubMed Web of Science 5. Yen , Z.C. , Meyer , I.M. , Karalic , S. , and Brown , C.J. ( 2007 ). A cross-species comparison of X-chromosome inactivation in Eutheria . Genomics 90 , 453 – 463 . doi: 10.1016/j.ygeno.2007.07.002 . OpenUrl CrossRef PubMed Web of Science 6. ↵ Wutz , A. , Rasmussen , T.P. , and Jaenisch , R. ( 2002 ). Chromosomal silencing and localization are mediated by different domains of Xist RNA . Nat Genet 30 , 167 – 174 . doi: 10.1038/ng820 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Migeon , B.R. , Kazi , E. , Haisley-Royster , C. , Hu , J. , Reeves , R. , Call , L. , Lawler , A. , Moore , C.S. , Morrison , H. , and Jeppesen , P. ( 1999 ). Human X inactivation center induces random X chromosome inactivation in male transgenic mice . Genomics 59 , 113 – 121 . doi: 10.1006/geno.1999.5861 . OpenUrl CrossRef PubMed Web of Science 8. ↵ Czermiński , J.T. , and Lawrence , J.B. ( 2020 ). Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation . Dev Cell 52 , 294 - 308 .e293. doi: 10.1016/j.devcel.2019.12.015 . OpenUrl CrossRef 9. ↵ Migeon , B.R. , Chowdhury , A.K. , Dunston , J.A. , and McIntosh , I. ( 2001 ). Identification of TSIX, encoding an RNA antisense to human XIST, reveals differences from its murine counterpart: implications for X inactivation . Am J Hum Genet 69 , 951 – 960 . doi: 10.1086/324022 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Migeon , B.R. , Lee , C.H. , Chowdhury , A.K. , and Carpenter , H. ( 2002 ). Species differences in TSIX/Tsix reveal the roles of these genes in X-chromosome inactivation . Am J Hum Genet 71 , 286 – 293 . doi: 10.1086/341605 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Migeon , B.R. , Pappas , K. , Stetten , G. , Trunca , C. , and Jacobs , P.A. ( 2008 ). X inactivation in triploidy and trisomy: the search for autosomal transfactors that choose the active X . Eur J Hum Genet 16 , 153 – 162 . doi: 10.1038/sj.ejhg.5201944 . OpenUrl CrossRef PubMed 12. Weaver , D.D. , and Gartler , S.M. ( 1975 ). Evidence for two active X chromosomes in a human XXY triploid . Humangenetik 28 , 39 – 42 . doi: 10.1007/BF00272480 . OpenUrl CrossRef PubMed 13. Jacobs , P.A. , Matsuyama , A.M. , Buchanan , I.M. , and Wilson , C. ( 1979 ). Late replicating X chromosomes in human triploidy . Am J Hum Genet 31 , 446 – 457 . OpenUrl PubMed Web of Science 14. ↵ Jacobs , P.A. , and Migeon , B.R. ( 1989 ). Studies of X-chromosome inactivation in trisomies . Cytogenet Cell Genet 50 , 75 – 77 . doi: 10.1159/000132727 . OpenUrl CrossRef PubMed Web of Science 15. Gartler , S.M. , Varadarajan , K.R. , Luo , P. , Norwood , T.H. , Canfield , T.K. , and Hansen , R.S. ( 2006 ). Abnormal X: autosome ratio, but normal X chromosome inactivation in human triploid cultures . BMC Genet 7 , 41 . doi: 10.1186/1471-2156-7-41 . OpenUrl CrossRef PubMed 16. Deng , X. , Nguyen , D.K. , Hansen , R.S. , Van Dyke , D.L. , Gartler , S.M. , and Disteche , C.M. ( 2009 ). Dosage regulation of the active X chromosome in human triploid cells . PLoS Genet 5 , e1000751 . doi: 10.1371/journal.pgen.1000751 . OpenUrl CrossRef PubMed 17. ↵ Migeon , B.R. , Sprenkle , J.A. , and Do , T.T. ( 1979 ). Stability of the “two active X” phenotype in triploid somatic cells . Cell 18 , 637 – 641 . doi: 10.1016/0092-8674(79)90118-1 . OpenUrl CrossRef PubMed Web of Science 18. ↵ Migeon , B.R. ( 2011 ). The single active X in human cells: evolutionary tinkering personified . Hum Genet 130 , 281 – 293 . doi: 10.1007/s00439-011-1016-7 . OpenUrl CrossRef PubMed 19. ↵ Migeon , B.R. , Beer , M.A. , and Bjornsson , H.T. ( 2017 ). Embryonic loss of human females with partial trisomy 19 identifies region critical for the single active X . PLoS One 12 , e0170403 . doi: 10.1371/journal.pone.0170403 . OpenUrl CrossRef PubMed 20. ↵ Migeon , B.R. ( 2017 ). Choosing the Active X: The Human Version of X Inactivation . Trends Genet 33 , 899 – 909 . doi: 10.1016/j.tig.2017.09.005 . OpenUrl CrossRef PubMed 21. ↵ Migeon , B.R. ( 2021 ). Stochastic gene expression and chromosome interactions in protecting the human active X from silencing by . Nucleus 12 , 1 – 5 . doi: 10.1080/19491034.2020.1850981 . OpenUrl CrossRef 22. ↵ Fukuda , A. , Tomikawa , J. , Miura , T. , Hata , K. , Nakabayashi , K. , Eggan , K. , Akutsu , H. , and Umezawa , A. ( 2014 ). The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice . Nat Commun 5 , 5464 . doi: 10.1038/ncomms6464 . OpenUrl CrossRef PubMed 23. ↵ Boulard , M. , Edwards , J.R. , and Bestor , T.H. ( 2016 ). Abnormal X chromosome inactivation and sex-specific gene dysregulation after ablation of FBXL10 . Epigenetics Chromatin 9 , 22 . doi: 10.1186/s13072-016-0069-1 . OpenUrl CrossRef PubMed 24. ↵ Okamoto , I. , Patrat , C. , Thépot , D. , Peynot , N. , Fauque , P. , Daniel , N. , Diabangouaya , P. , Wolf , J.P. , Renard , J.P. , Duranthon , V. , and Heard , E. ( 2011 ). Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development . Nature 472 , 370 – 374 . doi: 10.1038/nature09872 . OpenUrl CrossRef PubMed Web of Science 25. ↵ Petropoulos , S. , Edsgärd , D. , Reinius , B. , Deng , Q. , Panula , S.P. , Codeluppi , S. , Reyes , A.P. , Linnarsson , S. , Sandberg , R. , and Lanner , F. ( 2016 ). Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos . Cell 167 , 285 . doi: 10.1016/j.cell.2016.08.009 . OpenUrl CrossRef PubMed 26. ↵ Yan , L. , Yang , M. , Guo , H. , Yang , L. , Wu , J. , Li , R. , Liu , P. , Lian , Y. , Zheng , X. , Yan , J. , et al. ( 2013 ). Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells . Nat Struct Mol Biol 20 , 1131 – 1139 . doi: 10.1038/nsmb.2660 . OpenUrl CrossRef PubMed 27. ↵ Jiang , Z. , Sun , J. , Dong , H. , Luo , O. , Zheng , X. , Obergfell , C. , Tang , Y. , Bi , J. , O’Neill , R. , Ruan , Y. , et al. ( 2014 ). Transcriptional profiles of bovine in vivo pre-implantation development . BMC Genomics 15 , 756 . doi: 10.1186/1471-2164-15-756 . OpenUrl CrossRef PubMed 28. ↵ Migeon , B.R. ( 2019 ). The Non-random Location of Autosomal Genes That Participate in X Inactivation . Front Cell Dev Biol 7 , 144 . doi: 10.3389/fcell.2019.00144 . OpenUrl CrossRef 29. ↵ Moreira de Mello , J.C. , Fernandes , G.R. , Vibranovski , M.D. , and Pereira , L.V. ( 2017 ). Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing . Sci Rep 7 , 10794 . doi: 10.1038/s41598-017-11044-z . OpenUrl CrossRef PubMed 30. ↵ Brinkhof , B. , van Tol , H.T. , Groot Koerkamp , M.J. , Wubbolts , R.W. , Haagsman , H.P. , and Roelen , B.A. ( 2017 ). Characterization of bovine embryos cultured under conditions appropriate for sustaining human naï ve pluripotency . PLoS One 12 , e0172920 . doi: 10.1371/journal.pone.0172920 . OpenUrl CrossRef 31. ↵ Blakeley , P. , Fogarty , N.M. , del Valle , I. , Wamaitha , S.E. , Hu , T.X. , Elder , K. , Snell , P. , Christie , L. , Robson , P. , and Niakan , K.K. ( 2015 ). Defining the three cell lineages of the human blastocyst by single-cell RNA-seq . Development 142 , 3151 – 3165 . doi: 10.1242/dev.123547 . OpenUrl Abstract / FREE Full Text 32. ↵ Patrat , C. , Ouimette , J.F. , and Rougeulle , C. ( 2020 ). X chromosome inactivation in human development . Development 147 . doi: 10.1242/dev.183095 . OpenUrl Abstract / FREE Full Text 33. ↵ Zernicka-Goetz , M. , Morris , S.A. , and Bruce , A.W. ( 2009 ). Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo . Nat Rev Genet 10 , 467 – 477 . doi: 10.1038/nrg2564 . OpenUrl CrossRef PubMed Web of Science 34. ↵ Onichtchouk , D. , and Driever , W. ( 2016 ). Zygotic Genome Activators, Developmental Timing, and Pluripotency . Curr Top Dev Biol 116 , 273 – 297 . doi: 10.1016/bs.ctdb.2015.12.004 . OpenUrl CrossRef 35. ↵ Chitiashvili , T. , Dror , I. , Kim , R. , Hsu , F.M. , Chaudhari , R. , Pandolfi , E. , Chen , D. , Liebscher , S. , Schenke-Layland , K. , Plath , K. , and Clark , A. ( 2020 ). Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation . Nat Cell Biol 22 , 1436 – 1446 . doi: 10.1038/s41556-020-00607-4 . OpenUrl CrossRef 36. ↵ Motosugi , N. , Okada , C. , Sugiyama , A. , Kawasaki , T. , Kimura , M. , Shiina , T. , Umezawa , A. , Akutsu , H. , and Fukuda , A. ( 2021 ). Deletion of lncRNA XACT does not change expression dosage of X-linked genes, but affects differentiation potential in hPSCs . Cell Rep 35 , 109222 . doi: 10.1016/j.celrep.2021.109222 . OpenUrl CrossRef 37. ↵ Theunissen , T.W. , and Jaenisch , R. ( 2017 ). Mechanisms of gene regulation in human embryos and pluripotent stem cells . Development 144 , 4496 – 4509 . doi: 10.1242/dev.157404 . OpenUrl Abstract / FREE Full Text 38. ↵ Bermejo-Alvarez , P. , Ramos-Ibeas , P. , and Gutierrez-Adan , A. ( 2012 ). Solving the “X” in embryos and stem cells . Stem Cells Dev 21 , 1215 – 1224 . doi: 10.1089/scd.2011.0685 . OpenUrl CrossRef PubMed 39. ↵ Panning , B. , and Jaenisch , R. ( 1996 ). DNA hypomethylation can activate Xist expression and silence X-linked genes . Genes Dev 10 , 1991 – 2002 . doi: 10.1101/gad.10.16.1991 . OpenUrl Abstract / FREE Full Text 40. ↵ Datar , K.V. , Dreyfuss , G. , and Swanson , M.S. ( 1993 ). The human hnRNP M proteins: identification of a methionine/arginine-rich repeat motif in ribonucleoproteins . Nucleic Acids Res 21 , 439 – 446 . doi: 10.1093/nar/21.3.439 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Uysal , F. , Akkoyunlu , G. , and Ozturk , S. ( 2015 ). Dynamic expression of DNA methyltransferases (DNMTs) in oocytes and early embryos . Biochimie 116 , 103 – 113 . doi: 10.1016/j.biochi.2015.06.019 . OpenUrl CrossRef PubMed 42. ↵ McGraw , S. , Oakes , C.C. , Martel , J. , Cirio , M.C. , de Zeeuw , P. , Mak , W. , Plass , C. , Bartolomei , M.S. , Chaillet , J.R. , and Trasler , J.M. ( 2013 ). Loss of DNMT1o disrupts imprinted X chromosome inactivation and accentuates placental defects in females . PLoS Genet 9 , e1003873 . doi: 10.1371/journal.pgen.1003873 . OpenUrl CrossRef PubMed 43. ↵ Debril , M.B. , Dubuquoy , L. , Feige , J.N. , Wahli , W. , Desvergne , B. , Auwerx , J. , and Gelman , L. ( 2005 ). Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity . J Mol Endocrinol 35 , 503 – 517 . doi: 10.1677/jme.1.01856 . OpenUrl Abstract / FREE Full Text 44. ↵ Strehle , M. , and Guttman , M. ( 2020 ). Xist drives spatial compartmentalization of DNA and protein to orchestrate initiation and maintenance of X inactivation . Curr Opin Cell Biol 64 , 139 – 147 . doi: 10.1016/j.ceb.2020.04.009 . OpenUrl CrossRef 45. ↵ Goossens , K. , Van Poucke , M. , Van Soom , A. , Vandesompele , J. , Van Zeveren , A. , and Peelman , L.J. ( 2005 ). Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos . BMC Dev Biol 5 , 27 . doi: 10.1186/1471-213x-5-27 . OpenUrl CrossRef PubMed Back to top Previous Next Posted October 07, 2021. 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