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Differentially methylated CpG island within human XIST mediates alternative P2 transcription and YY1… Chapman, Andrew G; Cotton, Allison M; Kelsey, Angela D; Brown, Carolyn J Sep 9, 2014

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RESEARCH ARTICLECeanmalian females allowing for dosage compensation of expression.Chapman et al. BMC Genetics 2014, 15:89http://www.biomedcentral.com/1471-2156/15/89ing indirect evidence for conservation of P2 activity [11].In both mice and humans this region is contained withinDepartment of Medical Genetics, Molecular Epigenetics Group, Life SciencesCenter, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, CanadaX-linked genes between the sexes [1]. XCI is controlled byan alternatively spliced, long non-coding RNA calledXIST/Xist [2,3] that is up-regulated at the onset of XCIand is necessary for silencing [4-6]. XIST/Xist coats the fu-ture inactive X chromosome (Xi) [7] and recruits chroma-tin modifications that enable transcriptional silencing ofthe majority of genes on the Xi. Therefore, to achieveXIST/Xist regulation is dependent on promoter activitybut very little is known about the mechanisms controllingXIST/Xist promoter action beyond the minimal promotersequences required for transcription [8,9]. An alternativepromoter for mouse Xist called P2 has been observed andis located ~1500 bp downstream of the canonical pro-moter (P1) [10]. In humans, a region homologous to P2,in combination with P1, showed higher expression of a re-porter gene relative to clones containing P1 alone, provid-* Correspondence: Carolyn.Brown@ubc.caX-chromosome inactivation (XCI) is a process in mam-Background: X-chromosome inactivation silences one X chromosome in females to achieve dosage compensationwith the single X chromosome in males. While most genes are silenced on the inactive X chromosome, the genefor the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive Xchromosome with which the XIST RNA associates, triggering silencing of the chromosome. In mouse, an alternativeXist promoter, P2 is also the site of YY1 binding, which has been shown to serve as a tether between the Xist RNAand the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xistactivation, including absence of a functional antisense regulator Tsix, and absence of strictly paternal inactivation inextraembryonic tissues, prompting us to examine regulatory regions for the human XIST gene.Results: We demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive Xchromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island thatis differentially methylated between males and females and overlaps conserved YY1 binding sites that are onlybound on the inactive X chromosome where the sites are unmethylated. However, YY1 binding is insufficient todrive P2 expression or establish the DHS, which may require a development-specific factor. Furthermore, reductionof YY1 reduces XIST transcription in addition to causing delocalization of XIST.Conclusions: The differentially methylated DNase hypersensitive site within XIST marks the location of analternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are criticalfor silencing. In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1untethers the XIST RNA as well as decreasing transcription of XIST.Keywords: XIST, X-chromosome inactivation, Alternative promoter, YY1, DNA methylation, Long non-coding RNA,RNA FISH, DNase hypersensitivity siteBackground XCI, a female cell must ensure monoallelic up-regulationof XIST/Xist while a male cell must fully repress XIST/XistDifferentially methylatedhuman XIST mediates altand YY1 bindingAndrew G Chapman, Allison M Cotton, Angela D KelseyAbstract© 2014 Chapman et al.; licensee BioMed CentCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.Open AccesspG island withinrnative P2 transcriptiond Carolyn J Brown*ral Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Chapman et al. BMC Genetics 2014, 15:89 Page 2 of 11http://www.biomedcentral.com/1471-2156/15/89a CpG island that becomes methylated on the Xa to main-tain XIST repression [12]. Demethylation of this island iscorrelated with reactivation of XIST in human [13] andmouse [14] somatic cells. Moreover, the transcriptionfactor YY1 has recently been shown to be central to thecis specificity of Xist by tethering Xist RNA to the Xistgenomic locus at binding sites located within the XistCpG island [15].Beyond the XIST/Xist promoter, the minimal regionrequired for XCI, the X-inactivation centre, (XIC) [16,17],is believed to contain the regulatory elements essential forproper regulation of XIST/Xist. An invaluable tool inunderstanding Xist regulation has been mouse embryonicstem (ES) cells due to their ability to undergo XCI duringcellular differentiation [18]. Studies in mouse ES cells haverevealed a network of activating or repressing factors thatare either contained within the Xic or act through the Xic.Importantly, a non-coding RNA antisense to Xist, calledTsix, has been proven to be a crucial repressor of Xistduring cellular differentiation, blocking Xist on the Xa andbecoming downregulated on the Xi to allow Xist expres-sion [19,20]. Several other regulatory factors also act atthe XIC, including CTCF, which represses Xist upregula-tion [21,22], and RNF12 [23] and the non-coding RNAsJpx [22,24] and Ftx [25], which activate Xist expression.In humans, ES cells show considerable variationbetween lines and culture conditions in terms of theirXCI status [26-28]. The variability in human ES cells hashindered studies of XIST regulation but several lines ofevidence suggest that human and mouse XIST/Xistregulation may be different. The initial XCI in mouse ispaternally imprinted, with extraembryonic tissues main-taining inactivation of the paternal X. In humans, XCI israndom in all tissues with respect to the X chromosomethat undergoes inactivation, and the timing of the ini-tiation and role of any parental imprint has not beenresolved ([29] and reviewed in [30]). Thus the extent towhich the molecular machinery detailed for the initialchoice of X to inactivate in mice is functional in otherspecies remains to be determined. TSIX/Tsix has under-gone substantial divergence between species [31,32]. Inthe limited human cells reported to transcribe TSIX, theantisense RNA is not capable of repressing XIST nordoes transcription extend across the XIST promoter[32], a property crucial to Tsix repression of Xist inmouse [33], and fluorescent in situ hybridization (FISH)of TSIX transcripts indicates transcription is originat-ing from the Xi rather than the Xa [34]. In additionto differences in TSIX/Tsix, the entire XIC/Xic ispoorly conserved, suggesting that cis-regulatory elementsbetween species may be rapidly evolving [30,31], and Xist/XIST appears to be specific to eutheria with a differentlong non-coding RNA, Rsx, sharing similar properties inmarsupials [35].DNase I hypersensitivity (DHS) is commonly used toidentify regulatory elements due to their open chromatinstructure being sensitive to digestion by DNase I. Withinthe XIC, DHS mapping has revealed three putative re-gulatory elements, one downstream of XIST and twoupstream of XIST, that do not appear to be conservedbetween humans and mice [36]. In this paper we investi-gate a strong DHS site located within the human XISTCpG island and find that hypersensitivity correlates withP2 promoter usage and is independent of YY1 binding.ResultsDNase Hypersensitivity Site (DHS) mappingTo locate candidate cis-regulatory elements for XIST wesurveyed the XIC for DHS sites identified by genome-wide DHS-seq studies. Interestingly, the strongest DHSsites within the XIC are located within the XIST CpGisland, ~1.4 kb downstream of the XIST promoter, and arefemale-specific. We carried out a DNase I hypersensitivityassay at two regions, DHS 200b.1, which lies within theCpG island, and DHS 200a.1 which lies 518 bp down-stream of the CpG island (Figure 1A). DHS 200b.1 washypersensitive to digestion by increasing concentrations ofDNase I in female lymphoblast cells but not in malelymphoblast cells, while DHS 200a.1 was not hypersensi-tive in female or male lymphoblast cells (Figure 1B). Thedifference between male and female sensitivity at DHS200b.1 hinted at an Xi to Xa difference so we used Xi andXa-containing mouse-human hybrid cells to assess thechromatin of the Xa and Xi separately. We saw a signifi-cant increase in sensitivity at DHS 200b.1 in an Xi-containing mouse-human hybrid cell line and modestsensitivity at DHS 200a.1, while no effect of DNase treat-ment was seen at either region in an Xa hybrid cell line(Figure 1B) which demonstrated that the DHS site wasspecific to the Xi. The difference in sensitivity at regionDHS 200a.1 between female lymphoblast cells and Xi-containing mouse-human hybrid cells is likely a reflectionof the Xa in female cells masking weak sensitivity in theregion. Interestingly, HT-1080 male fibrosarcoma cellstransfected with a DOX inducible XIST cDNA clone(HT1080XISTi) [37] showed no hypersensitivity before orafter DOX induction of the full-length XIST transcript ateither DHS 200b.1 or DHS 200a.1 (Figure 1C). This maymean that establishment of the hypersensitivity within theCpG island of XIST occurs developmentally and is notrecapitulated in differentiated somatic cells.Promoter Activity of DHS 200b.1Genome-wide ChIP-seq data on the UCSC genome brow-ser shows peaks for several promoter-associated proteinsand chromatin modifications in female cell lines overlap-ping the XIST CpG island and DHS 200b.1. These marksinclude RNA Polymerase II, H3K4me3, H3K27ac, H3K9acChapman et al. BMC Genetics 2014, 15:89 Page 3 of 11http://www.biomedcentral.com/1471-2156/15/89[38] and over a dozen transcription factors includingYY1, a protein implicated in regulation of XCI in mouse[21]. Moreover, chromatin state segmentation studiesidentify the DHS 200b.1 region as an active promoter [39](Figure 2A) and studies in mouse have suggested thepresence of an alternative promoter approximately 1.5 kbwithin the Xist gene [10]. To address the question ofpromoter activity for DHS 200b.1 we performed 5′ RapidFigure 1 Xi-specific DNase I hypersensitive site ~1.5 kb within XIST. Awith locations of primers used for qPCR-based DHS assay. Shading of barsbased DHS assay on female and male lymphoblasts, Xi and Xa mouse-humDOX-inducible XIST transgene (HT1080XISTi). Sensitivity of biological triplicais used as a positive control. *p = 0.05-0.01, **p = 0.01-0.001, ***p < 0.001, nAmplification of cDNA Ends (RACE) assays on a regionoverlapping the hypersensitive site and the CpG islandof XIST (Figure 2A). 5′ RACE uncovered three transcrip-tion start sites (TSS) in the sense orientation spanning aregion of 404 bp across the CpG island (black arrows inFigure 2A). We therefore conclude that there is an alter-native promoter of human XIST that we designate as P2.In the antisense orientation a TSS located at the 3′ edge) Schematic indicating DHS sites from ENCODE genome-wide surveyreflects increasing amounts (0, 10, 20 or 40 U) of DNase. B) qPCR-an hybrids and an HT1080 (male fibrosarcoma) cell line containing ates ± standard deviation is shown relative to an unsensitive region. JPX.s. = not significant.Chapman et al. BMC Genetics 2014, 15:89 Page 4 of 11http://www.biomedcentral.com/1471-2156/15/89of the CpG island was also found, which we designateP2as (Figure 2B). Using qPCR primers across XIST exon 1no significant increase in transcription was found down-stream of P2, which is corroborated by RNA-seq data infemale cells [38], suggesting that P2 is unlikely to be thedominant XIST promoter (Figure 2C). Similarly, usingstrand-specific qRT-PCR we found P2as transcripts toFigure 2 Xi-specific DHS site correlates with an actively transcribing Pprimers used for qRT-PCR analysis. The outlined boxes are UCSC tracks showindata (upper) and RNA-seq data for the region (lower) [38,39]. B) Sequenced 5of XIST upstream and downstream of P2 determined by qRT-PCR (shown as bP2as transcripts relative to sense XIST (average of biological triplicates ± standbe at levels 0.001% of sense XIST transcripts, redu-cing the likelihood of these having a regulatory function(Figure 2D). Since XCI and the bulk of XIST regulationmust occur early in human development we also exam-ined the male human ES cell line CA1S for P2 or P2asfunction, which allows for examination of negative re-gulators of XIST and avoids the variable XCI patterns2 promoter. A) Schematic of XIST exon 1 indicating the locations ofg chromatin state segmentation as determined by compiled ChIP-seq‘RACE products with arrows indicating transcription orientation. C) Levelsiological triplicates ± standard deviation). D) Strand-specific qRT-PCR ofard deviation).in males [41]. To more completely assess the methylation-Control P2 HCFC1-Control P2 HCFC1-Control P2 HCFC1-Control P2 HCFC1IgG YY1AB02040608019 18 200d.2 qXIST5 YY10.150.100.050.000.100.200.00       Percent of RNA relative to EGFP siRNA1.501.000.500.001.501.000.500.00%Input%InputXi hybridCXa hybridFemale HT1080 XISTiNo DOXYY1XIST / DAPI / CoT-1LipoXIST / DAPI / CoT-1Figure 3 YY1 binding to region of P2 promoter is Xi-specific andupregulates P1 transcription. A) Chromatin immunoprecipitation(ChIP) of YY1 binding to P2 shown relative to input, at P2, a negativecontrol region within XIST, and a positive control region (HCFC1 genepromoter) for female (IMR-90), HT1080XISTi transgenic XIST, and Xi andXa-containing hybrids. A representative ChIP is shown ± standarddeviation of q-PCR triplicates; with replicate experiments showing thesame pattern but variable levels of IP relative to input. B) Q-PCRfollowing 72 h of siRNA mediated knockdown of YY1 in IMR-90 femalecells demonstrates a decrease in expression of XIST, both upstream(19, 18) and downstream of P2 (200d.2, qXIST5) relative to cells with acontrol (EGFP) knockdown. Levels of YY1 dropped to less than 20%confirming successful knockdown. C) FISH for XIST RNA following YY1knockdown. Left panel shows representative IMR-90 female cellstreated with lipofectamine alone (Lipo) and right panel shows cellsafter YY1 knockdown. Arrows indicate location of XIST RNA signal(green) which was substantially reduced after YY1 knockdown.Chapman et al. BMC Genetics 2014, 15:89 Page 5 of 11http://www.biomedcentral.com/1471-2156/15/89in female ES cells. We found transcription throughoutthe whole gene body of XIST and between 8683 bp to9934 bp beyond the 3′ end of XIST (Additional file 1:Figure S1A,B). Strand-specific RT-PCR assays determinedthat transcription is in antisense orientation, providing thefirst evidence for antisense transcription reaching theXIST promoter in humans (Additional file 1: Figure S1C).The faint RT-PCR bands, however, suggested low-leveltranscription, which was verified by qRT-PCR, indicatingthat the antisense transcript in this region is at 0.00012%of XIST expression levels in a somatic female lymphoblast.The region between as well as 5′ of these two primerscontains an enrichment of endogenous retrovirus in theantisense orientation and an Alu element so it is possiblethat transcription is initiating from an endogenous retro-viral long terminal repeat promoter.YY1 binding at the P2 regionWe next asked whether P2 activity and hypersensitivity atDHS 200b.1 was correlated with binding of YY1 in vivousing chromatin immunoprecipitation (ChIP). YY1 wasfound to be enriched at P2 in both female cells and Xi-containing hybrid cells but not Xa-containing hybrid cells,indicating Xi-specific binding (Figure 3A). We also foundYY1 binding at P2 in inducible XIST HT1080XISTi cellsbefore induction by DOX, despite these cells lacking aDHS, demonstrating that YY1 binding is insufficient toestablish hypersensitivity in the P2 region. Since YY1binding appeared to be independent of the DHS asso-ciated with P2 we wished to address the impact ofdown-regulating YY1 on the transcription of XIST. After72 h of siRNA-mediated knockdown of YY1 in IMR90female cells we saw a 50% decrease in expression of XIST,both upstream and downstream of P2, suggesting thatYY1 may be a regulator of transcription at P1 (Figure 3B).By fluorescent in situ hybridization for the XIST RNA weobserved only a small focus of expression in 41/86 (48%)of cells and no detectable signal in 35/86 (40%) of cells,with substantial delocalization of the RNA in theremaining 12% of cells (Figure 3C).DNA Methylation at P2 CpG islandAs diagrammed in Figure 2, the region around P2 isclassified as a CpG island according to the more relaxed‘intermediate’ classification that requires GC contentgreater than 50% and an observed CpG frequency toexpected CpG frequency of greater than 0.48 over at least200 bp [40]. The region 5′ to the CpG island has beenpreviously reported to be differentially methylated byrestriction enzyme analysis [12] and both the regionimmediately upstream of the XIST major promoter (P1 –two sites) and three sites within the P2 island (see Figure 4)are present on the Illumina 450 K bead chip and showintermediate methylation in females and hypermethylationacross this region, including across the differentiallybound YY1 sites, we designed four pyrosequencing assays25%50%75%100%DNA methylationCpG islandXISTP1P2asP2A repeatshewotionlumChapman et al. BMC Genetics 2014, 15:89 Page 6 of 11http://www.biomedcentral.com/1471-2156/15/89to analyse 18/21 CpG dinucleotides in the CpG island asshown in Figure 4. In general, male samples or Xa-containing hybrids showed over 60% methylation,while the Xi-containing hybrid had low methylation (seealso Table 1). Females, and also the HT1080XISTimale cell line with a single copy XIST transgene, showed0%73,071,444chrXCpGYY1Pyro assay450K assayA BFigure 4 DNA methylation of the CpG island containing XIST P2. Sc5′ A repeats. Methylation levels shown for each site are the average of thybrids, and the HT1080 with XIST transgenic line. Shown below is the locaat 18 of the 23 CpG sites in the intermediate density CpG island. Also showhttp://www.uniprot.org/uniprot/P25490) as well as the CpGs analysed by Ilintermediate methylation.DiscussionDHS sites serve as a molecular mark to identify impor-tant regulatory elements, and a strong DHS within thehuman XIST gene has been shown to be female-specific[42], suggesting an important regulatory role in X in-activation. Human ES cells have not provided as tract-able a model for the study of human XIST regulation asmouse ES cells have been for the study of the earlyTable 1 Activities observed at XIST P2 regionDHS@ +P2 activity ++YY1 binding +++DNAmethylationMale - - - 78%Female + + + 44%Xa Hybrid - -# - 83%Xi Hybrid + +* + 7%HT1080XISTi - - + 39%Male hES -** - -** 80%**@DHS: DNase Hypersensitive Site (from Figure 1; except for male hES (**)from UCSC).+P2 activity from RACE in Figure 2 or #[2]. *band observed with RACE, butnot sequence-confirmed.++YY1 binding from Figure 3, except ** from UCSC.+++DNA methylation is an average from 18 sites as shown in Figure 4.events of XCI in mouse. Therefore, we have assessed therole of the regulatory region demarcated by the DHS sitethrough correlation of features in a variety of model celltypes, as summarized in Table 1. The female-specificity ofthe DHS can be attributed to the presence of an Xi, asshown by the analysis of the somatic cell hybrids. The500bp73,070,847C DmaleXa hybridsfemaleXi hybridsHT1080 XISTimatic of the 5′ end of XIST showing P1 and P2 with the interveningmale cell lines, three female cell lines, two Xa and four Xi-containingn of the pyrosequencing assays (A to D) designed to assess methylationare the YY1 sites (based on consensus sequence 5′-CCGCCATNTT-3′ fromina 450 K Bead Chip.chromatin marks for this region suggest the presence ofpromoter activity, and we verified that ′P2′ is the originfor a variety of transcripts. Presence of the DHS was com-pletely concordant with P2 promoter activity; however, inmouse, the P2 region has also been shown to be the site ofbinding of YY1. Therefore we assessed YY1 binding byChIP and confirm that YY1 binds to the human P2region. YY1 binds the transgenic XIST, which was inte-grated in somatic cells, and therefore YY1 binding doesnot require passage of the DNA through development.Furthermore, in these HT1080-derived transgenic cells,no evidence for P2 transcriptional activity is observed([37], data not shown), separating P2 function fromYY1 binding. Therefore, P2 function is completelyconcordant with the DHS presence, and may requireadditional factors, that are perhaps acquired develop-mentally, for establishment. While the DHS within theXIST island is the only female-specific site near the XICregulatory region, two other female-specific DHS siteswere reported on the human X chromosome, and all wereassociated with long non-coding RNA loci [42]. The othertwo sites, FIRRE (LOC286467; [43]) and the noncodingRNA LOC550643 are also affiliated with complex tandemrepeats, differential methylation and CTCF binding, rem-iniscent of the DXZ4 region [44].Chapman et al. BMC Genetics 2014, 15:89 Page 7 of 11http://www.biomedcentral.com/1471-2156/15/89Both humans and mice demonstrate a P2 within thebody of XIST/Xist, and in both cases, transcription fromthis promoter would generate a transcript lacking the 5′A repeats that are critical for the silencing function ofXIST/Xist [37,45], raising the question of whether P2transcription might be secondary to another function forthe region. The first alternative role we considered wasthe generation of other transcripts. All transcription inthe region was only observed from the active XIST/Xistgene on the Xi, unlike some short RNAs that are tran-scribed from repressed polycomb target genes or im-printed loci [46]. In mice, there is a 1.6 kb transcript,called RepA, that is expressed from the active locus, andthus the future Xi; however, as the name suggests, thistranscript contains the A repeats [47]. We did identifyan antisense transcript; however, the very low abundancesuggests that this transcript is also not functional;although higher levels of such an antisense might inter-fere with P1 transcription, and therefore the sense P2transcript may be involved in preventing P2 antisensetranscription from silencing functional P1 transcription.Interestingly, we also observe an antisense transcriptacross XIST in human ES cells; however, again, thistranscript is at such low levels it seems unlikely toreflect a biologically relevant function.If generation of biologically active transcripts is notthe principal role of this region, transcription in theregion might be an indirect outcome of YY1 binding totether the RNA as YY1 binding has recently been shownto act as a ‘tether’ for the Xist RNA to bind to the DNAof the chromosome. YY1 binding in the region is seen tobe female-specific in browser tracks, and we confirmedthat this YY1 binding is Xi-specific as it is observed inXi, but not Xa-containing hybrids. Interestingly, in thesesomatic cell hybrids, despite binding of YY1, the RNA isnot tethered to the chromosome, and drifts away [48],implicating further species-specific factors in the abilityof DNA to interact with the cis-transcribed RNA. Wealso observed YY1 binding in HT1080XISTi transgeniccells; however, these cells fail to demonstrate P2 activity,suggesting that P2 transcription is not simply a functionof YY1 binding. To determine if YY1 binding impactedlocalization or transcription of XIST we knocked downYY1 with siRNA. Interestingly, we did observe a reduc-tion in XIST RNA abundance, across the gene, uponknock-down of YY1. This could reflect a role for YY1 intranscription from P1. An alternative explanation couldbe that delocalized XIST is more rapidly degraded; how-ever, in the mouse/human somatic cell hybrids the delo-calized XIST still had a half-life equivalent to localizedXIST [48]. There could also be downstream effects dueto the knockdown of the important YY1 protein thathave an indirect effect on XIST transcription; however,reporter assays have shown that inclusion of this regionaugments transcription [11], supporting an enhanceraction for this region.The binding of YY1 was seen to be concordant withlack of methylation of the CpG island within XIST. Thisregion is normally differentially methylated in females,and differentially methylated regions (DMRs) are oftenassociated with mono-allelic expression, similar to thatobserved for XIST, and also imprinted genes. Such DMRsoften overlap binding regions for the insulator/enhancerblocking factor CTCF [49], and there is CTCF bindingdownstream of the XIST DMR and upstream of P1; how-ever, neither is sex-specific [41] suggesting that they arenot primary regulators of XIST expression. While bindingof YY1 to the imprinted Peg3 gene was suggested to bemethylation-sensitive [50], the binding of YY1 to DXZ4 issimilarly restricted to the hypomethylated allele, howeverbinding in vitro is not blocked by DNA methylation [51].Thus, while YY1 binding is concordant with hypomethyla-tion, it is not clear wehther DNA methylation preventsYY1 binding to the Xa. Genome-wide analyses haveshown many additional transcription factors binding theP2 DMR region, some combination of which likelycontributes to activation of P2 transcription and establish-ment of the DHS, possibly during early development.ConclusionsWe report an Xi-specific DHS site within the human XISTlocus. This site overlaps a weak CpG island that showsdifferential methylation between the Xa and Xi. Thismethylation spans five consensus YY1 binding sites, and,consistent with ENCODE data, YY1 binds in a female-specific manner. The DHS is concordant with P2 pro-moter activity; however, transcription from the conservedP2 results in a transcript of unknown function that lacksthe A repeats previously shown to be critical for silencing.In addition to P2 activity, this region apparently functionsas both an enhancer and RNA tether, as knockdown ofYY1 reduces P1 transcription and also results in loss ofXIST localization to the Xi.MethodsDNase I hypersensitivity2,000,000 cells were harvested and lysed using 0.1% NP40in resuspension buffer (RSB) (10 mM Tris pH 7.4, 10 mMNaCl, 3 mM MgCl2). Nuclei were resuspended in RSBand digested with 0U, 10U, 20U or 40U of DNase I for10 min at 37°C. Digestion was stopped and DNA was ex-tracted using 0.8 mL of DNAzol (Invitrogen) followed byethanol precipitation. The DNA was diluted to a finalconcentration of 20 ng/μl, to be used in qPCR. Primersfor qPCR were designed to span the test hypersensitivesite (200b.1 and 200a.1) as well as a positive controlregion (JPX) and an insensitive region (XIST3′5′). Hyper-sensitivity was calculated by normalizing each DNase IChapman et al. BMC Genetics 2014, 15:89 Page 8 of 11http://www.biomedcentral.com/1471-2156/15/89concentration to the insensitive region and then resultsfrom each DNase I concentration were plotted as a folddifference from the untreated sample.Tissue culture and cell linesMouse-human somatic cell hybrid cell lines, t75-2maz34-1a (containing a human Xi) and t60-12 (containing ahuman Xa) [52] were cultured at 37°C in alpha Mini-mum Essential Medium (MEM) supplemented with 7.5%fetal calf serum (PAA Laboratories Inc), 1% penicillin/streptomycin (Life Technologies) and 1% L-glutamine(Life Technologies). The GM11200 and GM7057 malelymphoblast cell lines and GM11201, and GM7350female lymphoblast cells lines (Coriell cell repository)were maintained in Roswell Park Memorial Institute(RPMI) medium supplemented with 15% fetal calf serum(PAA Laboratories Inc), 1% penicillin/streptomycin (LifeTechnologies) and 1% L-glutamine (Life Technologies).The IMR90 female fibroblast cell line was cultured with10% fetal calf serum (PAA Laboratories Inc) and 1% L-glutamine (Life Technologies). The HT1080XISTi trans-genic cell line containing a DOX-inducible XIST wascultured in Dulbecco’s Modified Eagle Medium (DMEM)supplemented with 10% fetal calf serum (PAA LaboratoriesInc), 1% penicillin/streptomycin (Life Technologies) and1% L-glutamine (Life Technologies). CA1S cells were cul-tured as described [53] on Matrigel (BD Biosciences) coated6-well plates in mTeSR1 basal medium (STEMCELL)supplemented with mTeSR1 5x supplement (STEMCELL)and passaged using Accutase (STEMCELL). All lines werecultured at 37°C.PCR and quantitative PCRPCR was performed with 100 ng of genomic DNA tem-plate, 1U Taq polymerase, 0.2 mM dNTPs, 1.5 mMMgCl2, 1X PCR buffer (all from Invitrogen) and 0.5 μMof both forward and reverse primers. PCR was per-formed using 30–40 cycles of [95°C for 30 s, 55°-60°Cfor 30 s, 72°C for 1 min]. Quantitative PCR (qPCR) wasperformed using the StepOnePlusTM Real-Time PCRSystem (Applied Biosystems) and the qPCR reaction mixwas composed of 0.2 mM dNTP mix, 2.5 mM MgCl2,1X HS reaction buffer, 1X EvaGreen dye (Biotum),0.25 μM forward and reverse primer, and 0.8 U MaximaHot Start Taq (Fermentas) or AptaTaq Fast DNA poly-merase (Roche) and cycling conditions were as follows:95°C for 5 min, followed by 40 cycles of [95°C for 15 s,60°C for 30 s, 72°C for 1 min]. Each sample and negativecontrol was assayed in triplicate.RNA extraction and reverse transcriptionRNA was extracted using Trizol (Invitrogen) followingthe manufacturer’s instructions. RNA was treated usingthe DNA-free kit (Ambion) to remove genomic DNAcontamination according to the manufacturer’s instruc-tions and RNA concentrations were determined using aspectrophotometer. Reverse transcription (RT) of RNAwas carried out using 2 μg of RNA, 1x first strand buffer(Invitrogen), 0.01 mM Dithiothreitol (DTT) (Invitrogen),0.125 mM dNTPs, 1 μL random hexamers, 1 μl RNaseInhibitor (Fermentas) and 1 μL (1U) of Moloney MurineLeukemia Virus reverse transcriptase (M-MLV) and waterwas added to a total volume of 20 μL. Reactions withoutRT were also carried to out to test for complete removalof genomic DNA contamination. Reaction mixes were in-cubated for 1 h at 42°C and heat inactivated by incubatingat 95°C for 5 min.For strand-specific RT reactions 2 μg of RNA wasmixed with 0.50 μM dNTPs and 2 pmol of sense or anti-sense gene specific primer with a T7 sequence tag onthe 5′ end of the primer and then heated to 70°C for5 min. Following this incubation the tubes were placedon ice for 1 min and then mixed with 1x first strandbuffer (Invitrogen), 0.005 DTT, 1 μL (1U) RNase Inhibi-tor (Fermentas) and 1 μL (1U) Superscript III. Reactionsminus RT were also carried to out to test for completeremoval of genomic DNA contamination. Reaction mixeswere incubated for 1 h at 55°C and heat inactivated byincubating at 95°C for 5 min.5′ and 3′ rapid amplification of cDNA ends5′ RACE was performed using the First Choice RLM-RACE kit (Ambion) as per the manufacturer’s instruc-tions. 5′ RACE cDNA was amplified using nested PCRreactions (components as above) with 35 cycles eachand annealing temperatures of 57°C, for the outer PCRreaction, and 59°C for the inner PCR reaction. 3′ RACEwas performed using the First Choice RLM-RACE kit(Ambion) as per the manufacturer’s instructions. PCRproducts from both 5′ and 3′ RACE were analyzedusing 2% gel electrophoresis and PCR products purifiedwith the QIAquickGel extraction kit (Qiagen) forSanger sequencing.siRNA-mediated knockdownKnockdown was performed according to the manufac-turer’s instructions. Briefly, 100,000 cells were seededinto each well of a 24 well tissue culture plate. After24 hrs the cells were transfected with 2 μL of Dharmafect4 transfection reagent (Thermo Scientific) and 0.05 μM ofsiGenome SMARTpool siRNA (M-011796-02 for YY1)and harvested after a further 72 hrs.Chromatin immunoprecipitationBriefly, 5,000,000 cells were crosslinked using with 1%formaldehyde and lysed with cell lysis buffer (10 mMTris–HCl, pH 8, 10 mM NaCl, 3 mM MgCl2, 0.5% Igepal,protease inhibitor). Nuclei were resuspended in nuclearChapman et al. BMC Genetics 2014, 15:89 Page 9 of 11http://www.biomedcentral.com/1471-2156/15/89lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris–HCl,pH 8, protease inhibitor) and sonicated for 10 min to achromatin range of 150 bp to 500 bp. NaCl was added tothe chromatin to a final concentration of 150 mM andthen diluted in ChIP dilution buffer (0.01% SDS, 1.1%Triton X-100, 150 mM NaCl, 16.7 mM Tris–HCl pH 8).1% chromatin was collected for input DNA and remainingchromatin was incubated with either 10ug IgG antibody(I8140; Sigma) or 10 ug YY1 antibody (sc-1703; Santa CruzBiotechnology, Inc.) overnight. Chromatin and antibodywas incubated with blocked protein A:G beads and thenwashed with low-salt buffer (0.1% SDS, 1% Triton X-100,2 mM EDTA, 20 mM EDTA, 20 mM Tris–HCl, pH 8,150 mM NaCl), high salt buffer (0.1% SDS, 1%TritonX-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8, 500 mMNaCl), LiCL buffer (0.25 M LiCl, 1% Igepal, 1% Deoxycho-late, 1 mM EDTA, 10 mM Tris–HCl, pH 8) and TE buf-fer (10 mM EDTA, 10 mM Tris–HCl, ph 8) and theneluted with elution buffer (1% SDS, 0.1 M NaHC3).Precipitated chromatin and input DNA were was uncross-linked overnight in elution buffer and 0.192 M NaCl andproteinase K and purified using QIAquick DNA purifica-tion columns. Locus-specific enrichment was quantifiedusing qPCR.Statistical analysisStatistical analysis was performed using GraphPad Prism5.02. One-way ANOVA was used to test for significancein DHS experiments between different concentrations ofDNase I.FISH analysisRNA FISH was carried out as described by [54]. AnXIST probe and human Cot-1 DNA (Invitrogen) probewere fluorescently labelled by nick translation (AbbottMolecular Inc.) using SpectrumGreen-UTP (Vysis) forXIST and SpectrumRed-UTP (Vysis) for Cot-1. The XISTprobe used, HbC1a, covers a ~1.6 kb region of XIST in-cluding most of the repeat A region. Briefly, cells grownon glass coverslips were permeabilized in 0.5% TritonX-100 for eight minutes and then fixed in 4% para-formaldehyde (Electron Microscopy Sciences) for eightminutes. Cells were hybridized overnight with 150 ngXIST probe and 150 ng human Cot-1 DNA probe. Fol-lowing a series of post-hybridization rinses, the co-verslips were counterstained with DAPI and mountedon glass slides with Vectashield (Vector Laboratories)for imaging.DNA methylation analysis by pyrosequencingThree female cell lines (GM08399, GM05396 andGM08134), two male cell lines (GM7057 and GM1200),two Xa hybrids (t60-12 and AHA-11aB1) and four Xihybrids (t86-B1maz-1b, t75-, tHM-1A and tHM-34-2A)were each assessed for methylation using four pyrose-quencing assays. Pyrosequencing was performed using aPyromark ID machine as previously outlined in Cottonet al. [55]. Briefly, approximately 25 ng of bisulfiteconverted DNA was PCRed along with 1X PCR Buf-fer (Qiagen), 0.2 mM dNTPs, 0.025 U HotStart Taq DNAPolymerase (Qiagen), 0.25 mM forward primer and0.25 mM reverse primer (listed in Additional file 2:Table S1). PCR cycling conditions were the same forall four Pyrosequencing assays, 95°C for 15 min, 50 cyclesof 94°C for 30 s, 55°C for 30 s, 72°C for 60 s, followed by afinal step of 72°C for 10 min.Additional filesAdditional file 1: Figure S1. Transcription at the XIST locus in CA1Smale hES cells A) Schematic of XIST indicating primer positions (*) usedin RT-PCR. B) RT-PCR in CA1S cells at the XIST locus. C) Strand specificRT-PCR to determine orientation of transcription.Additional file 2: Table S1. Primers used in this manuscript.AbbreviationsXCI: X-chromosome inactivation; Xi: Inactive X chromosome; XIC: X-inactivationcenter; FISH: Fluorescent in situ hybridization.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAGC designed and carried out the studies described and drafted themanuscript. CJB participated in the study design, results interpretation andmanuscript writing. AMC designed and analysed the methylation assays andthe preparation of the figures, ADK performed the FISH analysis. All authorshave read and approved the final manuscript.AcknowledgementsThe authors thank Sarah Baldry for cell culture and laboratory assistance,Jordan Hendriksen for pyrosequencing and all members of the Brownlaboratory for helpful discussions. This work was supported by CIHRoperating grant MOP-13690 and AGC was supported by a CIHR studentship.We thank Dr. Andras Nagy, Dr. Janet Rossant, Marina Gertsenstein, KristinaVinterstein, Masha Mileikovsky and Jonathon Draper as well as Dr. J. Piret andlab for CA1S cells.Received: 16 May 2014 Accepted: 18 July 2014Published: 9 September 2014References1. Lyon MF: Gene action in the X-chromosome of the mouse (Mus musculus L.).Nature 1961, 190:372–373.2. 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BMC Genetics 2014 15:89.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionChapman et al. BMC Genetics 2014, 15:89 Page 11 of 11http://www.biomedcentral.com/1471-2156/15/89Submit your manuscript at www.biomedcentral.com/submit

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