Judit Balog

Dan Miller

Elena Sanchez-Curtailles

Jose Carbo-Marques

Gregory Block

Marco Potman

Peter De Knijff

Richard Jlf Lemmers

Stephen J Tapscott

Ph.D Silvère M Van Der Maarel

Epigenetic regulation of the X chromosomal macrosatellite repeat encoding for the cancer testis gene CT47

Keywords: Macrosatellite repeats, cancer-testis antigen, copy number variation, epigenetics, DNA methylation, histone modifications, D4Z4, DXZ4, FSHD Introduction

Macrosatellite repeats (MSRs) present an extreme example of copy number variation, yet their epigenetic regulation in normal and malignant cells is largely understudied.

The CT47 cancer-testis antigen located on human Xq24 is organized as an array of 4.8kb-large units. CT47 is expressed in testis and in certain types of cancer, but not in non-malignant somatic tissue. We used CT47 as a model to study a possible correlation between copy number variation, epigenetic regulation and transcription originating from MSRs in normal and malignant cells. In lymphoblastoid cell lines and in primary fibroblasts CT47 expression was absent, consistent with the observed heterochromatic structure and DNA hypermethylation of the CT47 promoter. Heterochromatinization of CT47 occurs early during development as human embryonic stem cells show high levels of DNA methylation and repressive chromatin modifications in the absence of CT47 expression. In small cell lung carcinoma cell lines with low levels of CT47 transcripts we observed reduced levels of H3K9me3 and H3K27me3 without concomitant increase in euchromatic histone modifications. DNA methylation levels in the promoter region of CT47 are also significantly reduced in these cells. This supports a model in which during oncogenic transformation there is a relative loss of repressive chromatin markers resulting in leaky expression of CT47. We conclude that some MSRs, like CT47 and the autosomal MSRs TAF11-Like, PRR20, ZAV and D4Z4, the latter being involved in facioscapulohumeral muscular dystrophy, seem to be governed by common regulatory mechanisms with their abundant expression mostly being restricted to the germ line.

which was recently suggested to be involved in a psychiatric disorder, and very recently SST1, PRR20 and ZAV 9 . D4Z4 is characterized by bidirectional transcriptional activity, high DNA methylation, and the presence of both heterochromatic histone modifications such as histone 3 lysine 9 trimethylation (H3K9me3) as well as the transcriptionally permissive histone modification H3K4me2. Upon contraction, as seen in FSHD, D4Z4 becomes hypomethylated 10 , shows selective loss of H3K9me3, with secondary losses of heterochromatin protein 1 gamma and the cohesin complex 11 , thus leading to a more open chromatin structure. DXZ4 has chromatin features very similar to D4Z4 with but shows opposite chromatin configurations on the active (Xa) and inactive (Xi) X chromosome. On Xa, DXZ4 is organized as constitutive heterochromatin with high levels of H3K9me3, while on the Xi, DXZ4 adopts a more euchromatic structure with high levels of H3K4me2 and H3K9ac. Also DXZ4 is bidirectionally transcribed with discrete differences in the transcriptional landscape on Xa and Xi [START_REF] Chadwick | DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts[END_REF] .

From these studies the picture emerges that CNV, expression profile and chromatin structures are important attributes of MSRs which, when disturbed may have consequences for genome integrity and function, as has recently been demonstrated in S. cerevisiae where large rDNA repeats are required for the maintenance of genome integrity 12 .

Cancer/testis antigens (CTAs) represent a class of genes that are typically only expressed in germ cells of the testis, fetal ovary and placenta, and sometimes in brain, and which also show expression in various tumor types [START_REF] Caballero | Cancer/testis (CT) antigens: potential targets for immunotherapy[END_REF][START_REF] Hofmann | Genome-wide analysis of cancer/testis gene expression[END_REF] . Epigenetic changes in cancer typically involve hypermethylation of CpG islands and hypomethylation of repetitive DNA elements [START_REF] Pfeifer | DNA methylation patterns in lung carcinomas[END_REF][START_REF] Smet | DNA hypomethylation in cancer: Epigenetic scars of a neoplastic journey[END_REF] . Hypermethylation can lead to transcriptional silencing of tumor suppressor genes while the effect of hypomethylation in cancer is less clear, but thought to contribute to genomic instability. In accordance, it has been established that many CTAs, show expression in malignancies which is associated with promoter demethylation [START_REF] Luetkens | Expression, epigenetic regulation, and humoral immunogenicity of cancer-testis antigens in chronic myeloid leukemia[END_REF][START_REF] Xiao | Identification of a novel human cancer/testis gene MAEL that is regulated by DNA methylation[END_REF] . However, by and large the chromatin structure of CTAs under normal and malignant conditions has not been studied in great detail.

The cancer/testis gene CT47 on Xq24 is arranged as a MSR with up to 17 copies of a putatitve protein coding gene ordered head-to-tail [START_REF] Chen | Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome[END_REF] . Being a CTA and a MSR, in this study we set out to examine the array length and chromatin structure of this MSR in CT47 expressing and non-expressing cells.

Material and methods

Cell lines and culturing conditions

HapMap cell lines and primary foetal lung fibroblast WI38 were purchased from Coriell Cell Repositories and cultured according to the supplier instructions. SCLC cell lines were purchased from LGC Standards GmbH and cultured as described [START_REF] Carney | Establishment and identification of small cell lung cancer cell lines having classic and variant features[END_REF] , Human embryonic stem cells (hESC) WA9 and WA14 [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF] were purchased from WiCell Research Institute, Madison, Wisconsin, USA. Cells were cultured according to a published protocol [START_REF] Ware | A comparison of NIH-approved human ESC lines[END_REF] hESC and human embryonic body (hEB) samples were analysed for the presence of the differentiation markers NANOG , OCT4 and XIST as described [START_REF] Silva | X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells[END_REF] and showed that ESCs had differentiated properly.

Primers

All primers used in the study are described in Supplemental table 1. PCR cycling conditions were as follows: 15 minutes denaturation step at 94 ºC followed by 45 cycles of 94 ºC for 20 seconds, 55 ºC for 30 seconds and 72 ºC for 60 seconds and a final step of 72 ºC for 5 minutes. PCR products were sequenced and the level of methylation of individual CpGs was measured with ESME software (Epigenomics AG) [START_REF] Lewin | Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates[END_REF] .

Characterisation of the CT47 array

Chromatin immunopreciptation (ChIP)

Chromatin was prepared from cells fixed with 1% formaldehyde and then quenched with 125mM glycine. The ChIP procedure was done according to a published protocol 622) were purchased from Millipore. EZH2 antibody was purchased from Active Motif (#39103) and the histone H3 antibody (#ab1791) from Abcam.

To determine the level of nonspecific signals we used IgG, supplied with the antibodies for H3K9me3 and H3K27me3. Immunoprecipitation with histone antibodies and IgG was done with chromatin containing 3 ug DNA. Immunoprecipitation with EZH2 antibody and IgG was done with chromatin containing 6 ug DNA. The amount of immunopurified DNA was determined by quantitative PCR with specific primer pairs at three different sites in CT47: promoter primer pair (labeled A in Figure 1) exon 3 primer pair (labeled C in Figure 1), distal primer pair (labeled D in Figure 1). All primers were designed by primer3 program and optimized for minimizing sequence variations between individual repeat units. Primers were tested for specificity on a monochromosomal cell panel. A published primer pair was used for the GAPDH promoter [START_REF] Barrand | Promoter-exon relationship of H3 lysine 9, 27, 36 and 79 methylation on pluripotency-associated genes[END_REF] . Relative abundance of histone modifications were calculated in the following way. Enrichment values obtained with IgG were subtracted from the enrichment values from specific antibodies. Normalisation for copy number variation was done by correcting the enrichment values from the ChIP by the input values. Input values were obtained by determining the total amount of DNA used in the ChIP by using the specific primer pairs. These results are presented in the figures as relative enrichment to input. Samples were tested by ChIP in 2 independent experiments and every immunopurified DNA was quantified twice independently.

cDNA synthesis and quantification of CT47 mRNA

Human testis total RNA was purchased from Ambion (#AM7072). Total RNA was isolated from LCL cell lines, hESCs, hEBs from lines WA9 and WA14, and SCLC lines with Qiasol reagent (Qiagen #79306). RNA was purified using the miRNeasy kit (Qiagen #217004). cDNA synthesis was done with Omniscript Reverse transcription kit (Qiagen #205113) using 500ng total RNA according to the manufacturer instructions.

cDNA synthesis for all samples was done in parallel under identical conditions and repeated twice. cDNA was quantified by using specific intron spanning primers for CT47 using Sybergreen (BioRad #170-8882) mix and measurements were performed with MyiQ Single-Color Real-Time PCR Detection System, and analysed with the Bio-Rad iQ5 program. Samples were measured twice in duplicate.

Statistical analyses

Statistical analysis of DNA methylation levels of different samples was done by Mann-Whitney U test.

Results

No correlation between CT47 unit number and chromatin structure inside the CT47 array

HapMap cell lines were sized for the CT47 array located at Xq24 by PFGE analysis of EcoRI digested genomic DNA and using a probe recognizing the repeat (Supplemental figure 1). The CT47 repeat sizes of selected cell lines used in this study are listed in Table 1. Taking advantage of the hemizygosity of CT47 in males, to study the repeatsize dependency of the CT47 chromatin structure, four male samples were chosen with short CT47 arrays (4-7 repeat units) and two with long arrays (12 or 14 units). Female samples were selected based on their homozygosity for the CT47 repeat size. The relative abundance of dimethylated lysine 4 of histone H3 (H3K4me2), trimethylated lysine 27 of histone H3 (H3K27me3) and trimethylated lysine 9 of histone H3 (H3K9me3) in CT47 promoter region, exon 3 of the CT47 gene and 3700 base pairs distal of the array (Fig. 1) was determined by quantitative ChIP analysis. H3K4me2 is associated with an open chromatin structure, while H3K27me3 marks transcriptionally silenced regions and H3K9me3 is specific for heterochromatin. The GAPDH promoter region with an open chromatin structure was included as a control (Supplemental Fig. 2).

The most abundant histone modification at the CT47 promoter in the male cell lines is H3K9me3, suggesting that the chromatin is highly condensed in this region (Fig. 2a).

We also detected high levels of H3K27me3 and low levels of H3K4me2 further supporting a closed chromatin structure. Cell lines with short or long CT47 repeat arrays showed high inter-individual variation in H3K9me3 and H3K27me3 levels without clear correlation with repeat length. Distal to the array the relative abundance of H3K27me3 is higher and H3K9me3 levels are lower than in the CT47 promoter, while H3K4me2 levels are similarly low (Fig. 2b). There is no correlation between CT47 array length and chromatin structure distal to the array.

Similar to the male samples high levels of H3K9me3 and H3K27me3 and low levels of H3K4me2 were detected in the female cell lines with similarly sized long or short CT47 repeats on both alleles (Fig. 2c). The H3K9me3 levels were lower than or close to the H3K27me3 levels while in male samples H3K9me3 was the most abundant modification. Distal to the CT47 array the relative abundance of these histone modifications is similar as within the array in female samples (Fig. 2d). We also included a female primary fibroblast cell line (5-6 units) to exclude that immortalization by EBV has significant effects on the chromatin structure of CT47. We did not observe a difference in chromatin structure between female immortalized LCLs and female primary fibroblasts, excluding a large effect of the immortalization process. Thus, in normal somatic cells the CT47 repeat contains repressive chromatin markers and within the tested size range there is no correlation between CT47 repeat array size and chromatin structure.

CT47 is heterochromatic in human embryonic stem cells and embryonic bodies

Male WA14 and female WA9 human hESC cell lines were studied for CT47 chromatin structure in the pluripotent state and when differentiated into hEBs. This showed that H3K9me3 is already abundantly present on this locus in hESCs and in hEBs (Fig. 3).

H3K4me2 and H3K27me3 levels were equally low in these samples, which is different from LCLs or primary fibroblasts, where H3K27me3 was far more abundant then H3K4me2. We did not observe major changes in the relative abundance of the different histone markers during differentiation. We conclude that the CT47 promoter region is highly heterochromatic early in development and remains heterochromatic during early differentiation, suggesting that at these stages CT47 expression does not change.

Small cell lung carcinoma cells are expressing CT47 RNA

We selected 4 commercially available SCLC cell lines for determining CT47 expression in relation to chromatin structure. Two of them were already reported to express CT47 (NCI-H82 and NCI-H187) [START_REF] Chen | Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome[END_REF] while the 2 other SCLC cell lines were not studied yet (NCI-H69 and H1607). Human testis total RNA was included as a control. By quantitative RT-PCR we detected CT47 expression in all SCLC cell lines but not in hESCs. hEBs or in LCLs and fibroblast cell cultures studied. In good agreement with 1 2 previously published data, SCLC cell lines only express 0.1-3% of CT47 levels compared to testis (Fig. 4a) [START_REF] Chen | Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome[END_REF] .

Chromatin structure of CT47 array in SCLCs

We studied the role in transcription of the chromatin structure of the CT47 promoter region in all 4 SCLC cell lines. Figure 4c shows that the relative abundance of H3K4me2 is lower in SCLCs compared to LCL 385. However, H3K9me3 levels are dramatically reduced in all SCLC cell lines. Of all SCLCs, the highest levels of H3K9me3 are observed in sample NCI-H82, which expresses low levels of CT47.

SCLC cell lines NCI-H69, NCI-H187 and H1607 show similar low levels of H3K9me3.

H3K27me3 levels do not show a reduction but an increase except for NCI-H69.

H3K27me3 is generated by the catalytic subunit of the PRC2 complex, the histone methyl transferase EZH2. Transcriptionally silenced regions often show the simultaneous presence of H3K27me3 and EZH2. We therefore also tested the presence of EZH2 on the CT47 promoter in LCLs and SCLCs and observed that EZH2 levels are strongly reduced in SCLCs when compared to LCLs.

The chromatin structure of the SCLCs was also analyzed in the third exon of the CT47 gene and 3700 base pairs distal to the CT 47 array (Fig. 4b). Chromatin markers showed a very similar picture in the third exon as in the promoter region except sample NCI-H82 where we detected H3K9me3 levels comparable to the LCL sample. We detected high levels of H3K9me3 and H3K27me3 compared to H3K4me2 in the LCL 385.

Interestingly in this region H3K27me3 was more abundant than H3K9me3. SCLCs showed a general loss of repressive chromatin markers in this region. EZH2 levels corresponding to the H3K27me3 levels were high in LCL 385 and lower in the SCLCs. Thus, in LCLs the CT47 promoter region contains both the transcriptional repressive histone modification H3K27me3 together with EZH2, and the heterochromatic histone modification H3K9me3. In SCLCs the heterochromatic H3K9me3 marker is dramatically reduced and it seems that the epigenetic regulation of transcription through the PRC2 complex is disturbed: while EZH2 levels are decreased in SCLCs the histone marker generated by PRC2, H3K27me3 is not reduced. Thus we observed that transcriptional activity inversely correlates with H3K9me3 levels and EZH2 occupation, rather than directly with H3K4me2 levels.

DNA methylation of CT47 promoter region in LCLs, embryonic stem cells and SCLCs

Expression of CTAs in different tumor samples was previously shown to correlate with DNA hypomethylation [START_REF] Xiao | Identification of a novel human cancer/testis gene MAEL that is regulated by DNA methylation[END_REF][START_REF] Glazer | Integrative discovery of epigenetically derepressed cancer testis antigens in NSCLC[END_REF][START_REF] Bai | Promoter demethylation mediates the expression of ZNF645, a novel cancer/testis gene[END_REF] . Using bisufite sequencing we measured the level of DNA methylation in the promoter region of CT47, from 275 base pairs upstream from the first exon, in LCLs and SCLCs (Fig. 4e), as well as hESCs and hEBs (Supplemental figure 3). The promoter region of CT47 contains 7 CpGs. Quantitative methylation analysis of these 7 CpGs showed that the promoter region of CT47 is highly methylated in LCLs, hESCs and hEBs. A significant difference was detected in DNA methylation between LCLs and SCLCs, with SCLCs showing decreased methylation levels at all 7 CpGs (p<0.01).

Discussion

On human Xq24 highly homologous copies of CT47 are organized into an MSR of tandemly arranged units of each 4.8 kb in size. This polymorphic MSR varies from 4-17 copies in length (manuscript in preparation and [START_REF] Chen | Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome[END_REF] ). As CT47 has a very specific expression profile, it is a good model to study the tissue-specific epigenetic regulation of MSRs. CTAs form a large family of protein coding sequences with specific expression pattern: they are expressed in testis and certain types of cancers. Our study presents the first detailed analysis of the chromatin structure of a CTA in nonmalignant and malignant cells and during early stages of development. Although biological processes leading to cancer should preferably studied on human tumor material, the established SCLCs represent a valuable and readily available tool for studying genetic [START_REF] Singh | A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival[END_REF] and epigenetic changes [START_REF] Coe | Gain of a region on 7p22.3, containing MAD1L1, is the most frequent event in small-cell lung cancer cell lines[END_REF] in cancer.

Our expression data are in a good agreement with earlier studies showing that CT47 expression is restricted to testis and malignant cells [START_REF] Chen | Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome[END_REF] , albeit that the expression in SCLCs is only a fraction of that observed in testis. We can not exclude the possibility that only a small proportion of SCLC cells in culture express CT47, analogous to the presence of small subpopulation of solid tumors 2 .

We did not observe expression of CT47 in hESC cells and hEBs. The CTAs MAGE-A, GAGE and NY-ESO-1 were previously shown not to be expressed in hESCs [START_REF] Gjerstorff | Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation[END_REF] , while MAGE-A2 and MAGE-A6 expression could be detected in 20 days old hEBs. Although we can not exclude the possibility that the absence of CT47 transcript in our hESC and hEB samples is the result of the epigenetic fluidity of human hESCs [START_REF] Silva | X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells[END_REF][START_REF] Hall | X-inactivation reveals epigenetic anomalies in most hESC but identifies sublines that initiate as expected[END_REF][START_REF] Shen | X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations[END_REF] , our results suggest that CT47 transcription is induced at later stages of development, possibly between the pluripotent stem cell and primordial germ cell stages, from which matured germ cells are developing.

Chromatin analysis of non-expressing cells showed high levels of the heterochromatic histone modifications H3K27me3 and H3K9me3, and relatively low levels of the euchromatic histone modification H3K4me2 in the promoter region of CT47. We could not detect a correlation between CT47 array length and its chromatin structure in analogy with FSHD, where contractions only below a threshold of 11 units result in D4Z4 chromatin relaxation 11 . Rather, we observed a high variation in the abundance of different histone modifications in samples with the same array lengths (figures 2 and 3).

As D4Z4 shows similar variability in levels of histone modifications with repeat lengths >11 units, it is likely that 4 units of CT47 is sufficient to ensure a heterochromatic chromatin environment in non-germline cells and opens up the possibility that each MSR needs a minimal, but for each MSR unique, number of repeat units to establish its appropriate chromatin structure.

We observed a relative higher abundance of H3K27me3 to H3K9me3 in female samples. The observation that females have relatively higher levels of H3K27me3 than males, suggests that repression of CT47 is differently established on Xa and Xi. Indeed, Xi in females can be more densely populated with H3K27me3 then Xa [START_REF] Marks | High-resolution analysis of epigenetic changes associated with X inactivation[END_REF] . Nevertheless, this is different from the X chromosomal MSR DXZ4 showing that two MSR on Xq can behave very differently in chromatin structure on the Xa and Xi.

A unique feature of the pluripotent state is the so called bivalent chromatin state defined by the presence of permissive (H3K4me3) and repressive (H3K27me3) histone modifications [START_REF] Bernstein | A bivalent chromatin structure marks key developmental genes in embryonic stem cells[END_REF] Bivalent states are found at promoter regions of genes expressed at low levels in hESCs that are associated with differentiation. We detected identical low levels of H3K4me2, H3K4me3 and H3K27me3 in hESCs and hEBs at the CT47 promoter region, suggesting that the product of this gene is not necessary for early development.

Repetitive elements were identified as targets of PRC1 and PRC2 complexes in the pluripotent state [START_REF] Leeb | Polycomb complexes act redundantly to repress genomic repeats and genes[END_REF] . Our results show that the MSR CT47 has, like D4Z4 11 a heterochromatic structure already at early stages of development, marked by abundant H3K9me3 levels and that this chromatin structure does not change during differentiation. The high levels of H3K9me3 suggest that repression of MSR is mainly governed by SUV39H1 and SUV39H2 and not so much by PRC complexes. SCLC cell lines, which do express CT47, show a disturbed chromatin structure of this MSR. In addition, male cell lines NCI-H69 and NCI-H187 harbor three arrays and NCI-H82 two arrays of CT47 MSR suggesting that they have become aneuploid for the X chromosome. We detected significantly lower levels of H3K9me3 in these cancer cell lines without a concomitant relative increase in the abundance of the transcriptionally permissive histone modification H3K4me2. This might explain the relatively low levels of CT47 expression in these cells. Rather than transcriptional reactivation, SCLCs seem to show a relative loss of transcriptional repression resulting in leaky expression of CT47. Due to technical limitations we can currently not exclude the possibility that individual units within an array are differently organized.

In contrast to H3K9me3, H3K27me3 levels are not significantly lower in SCLCs, while EZH2 levels are significantly decreased in these samples. This shows that SCLC cells have serious defects in coordinated epigenetic regulation of this MSR, consistent with reports of genome wide epigenetic changes in other malignancies [START_REF] Smet | DNA hypomethylation in cancer: Epigenetic scars of a neoplastic journey[END_REF] .

CTA gene expression is often correlated with DNA promoter hypomethylation. We observed that all of the CpG dinucleotides examined in the promoter region of CT47 show a significant decrease in DNA methylation in SCLCs compared to the otherwise high methylation levels in nonmalignant cells. Epigenetic factors like DNA methylation and histone modifications are acting in a coordinated way to establish normal development. Our study reveals that transcriptional regulation/derepression of CT47 is dominated by the loss of repressive markers such as DNA methylation and H3K9me3: SCLCs with significantly lower levels of DNA methylation and H3K9me3 at the promoter region are expressing the CT47, despite of the absence of open chromatin markers.

We observed striking commonalities between the autosomal MSR D4Z4 and the Xchromosomal MSR CT47. In normal somatic cells, in hESCs and in hEBs both MSRs are highly methylated and have high levels of repressive chromatin markers. In the adult body DUX4 [START_REF] Snider | Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene[END_REF] , encoded by D4Z4, and CT47 transcripts are highly abundant in testis and ovary. In disease conditions, like FSHD or cancer, there is a loss of DNA methylation and repressive chromatin markers, but no increase in permissive chromatin modifications, leading to low or leaky expression levels of the MSR encoded genes.

Like CT47 expression in SCLCs, evidence was presented that DUX4 is expressed in malignant adult and fetal human rhabdomyosarcoma cell lines [START_REF] Kowaljow | The DUX4 gene at the FSHD1A locus encodes a pro-apoptotic protein[END_REF] Numbers in brackets are corresponding to the number of CT47 units in the sample. Figure 2a shows that the euchromatic H3K4me2 mark has similar low levels in all samples.

H3K27me3 is considered a transcriptional repressive chromatin mark, and is also very similar in samples 248, 185, 135 with shorter CT47 arrays and shows more variation in samples with longer arrays. The heterochromatic mark H3K9me3 shows highest individual variation, independent of the array length. There is no correlation between chromatin structure and array length.

  CT47 array size was determined by pulsed field gel electrophoresis (PFGE) after digestion of agarose-embedded DNA with EcoRI. Hybridisation was performed with radioactively labeled CT47 probe recognizing the CT47 unit sequence (NT_011786.15), generated by PCR. Quantitative methylation analysis of bisulfite converted DNA Genomic DNA was treated with sodium bisulfite (EZ DNA methylation kit; Zymo Research #D5002). Bisulfite-converted DNA was PCR amplified in a final volume of 25 μl containing 1X PCR buffer supplied with the enzyme, 25 mmol of each dNTP, 10 pmol of bis CT47 primer pair and 0.1 units of HotStar Taq DNA polymerase (Qiagen #203203). Primers specific for the CT47 promoter region were designed with MethPrime to amplify the genomic region 270 base pairs upstream of exon 1 of CT47.

  Figure 1

Figure 1

 1 Figure 1 Schematic representation of CT47 macrosatellite array. The CT47 repeat array is localized on Xq24 at coordinates chrX: 119,893,246-119,948,579 according to hg18 assembly. The size of the array varies between 4-17 units, each unit being 4.8kb in size. Chromatin markers were studied in the promoter region of the CT47 gene encoded by each unit (exons are indicated in grey boxes). Genomic locations where relative abundance of different histone modifications were quantified are represented by the black line; A represents ChIP primer set for CT47 promoter, B represents distal primerset located 3,7 kb from the array, C represents ChIP primer set for CT47 exon 3, and D represents the bisulphate primer set.

Figure 2

 2 Figure 2 Relative abundance of histone modifications at the CT47 promoter a male, c female and distal region b in male d in female LCL samples from the HapMap panel.

  Figure2bshows the histone modification profile at 3701 bp distal of the CT47 array, coordinates chrX: 119,952,280-119,952,414 (hg18). H3K27me3 levels are higher than H3K9me3 and H3K4me2 levels are similarly low as in the promoter region of CT47.Relative abundance of different histone modifications at the CT47 promoter c and distal region d in female samples with low CT 47 unit number (237) and with high CT47 unit number (166 and 138) on both X chromosomes. WI 38 is a primary foetal lung fibroblast and was used in the study to show that immortalization does not lead to changes in chromatin structure of CT47 in LCLs. Histone profiles in female samples are similar to male samples in both regions studied, showing a transcriptionally repressive state marked by high levels of H3K9me3 in the promoter region and by high H3K27me3 in the distal region with low H3K4me2 levels. There is no correlation between array size and chromatin structure in these region in female samples. The histone modification landscape of primary fibroblast cell line WI38 is very similar to the LCL samples.

Figure 3

 3 Figure 3Relative abundance of different histone modifications at CT47 in human male (WA 14) and female (WA 9) pluripotent embryonic stem cells (hESCs) and in differentiated embryonic bodies hEBs). The relative abundance of H3K4me2, associated with transcriptionally permissive chromatin, is increasing during differentiation in male and female samples. The repressive chromatin mark H3K9me3 is detected at high levels at pluripotent state and slightly decreases after differentiation. H3K27me3, associated with transcriptional repression, is detected in pluripotent state and increasing during differentiation.

Figure 4 a

 4 Figure 4 a Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47

Table 1

 1 List of cell lines included in the study:

	HapMap	Lab identifier	Cell type	Gender	CT47 unit
	identifier				number
	GM18980	237	LCL	female	5, 5
	GM12044	166	LCL	female	10, 10
	GM10831	138	LCL	female	11, 8
		WI 38	Primary foetal	female	5, 6
			lung fibroblast
	GM18561	248	LCL	male	4
	GM12144	185	LCL	male	5
	GM10830	135	LCL	male	6
	GM19204	334	LCL	male	7
	GM18953	274	LCL	male	12
	GM18504	385	LCL	male	14
		WA9	hESC	female	Nd*
		WA14	hESC	male	Nd*
		NCI-H69	SCLC	male	9, 9, 5
		NCI-H82	SCLC	male	10, 8
		NCI-H187	SCLC	male	8, 8, 4
		H1607	SCLC	male	Nd*
	*Nd means: not determined

. Antibodies against H3K4me2 (#07-030), H3K9me3 (#17-625) and H3K27me3 (#17-

Acknowledgements

The authors thank Peter Thijssen for critical reading of the manuscript and Yvonne Krom with helping the DNA methylation analysis.

Funding

This work was supported by institutional funds from the LUMC, the Netherlands Organization for Scientific Research [NWO 917.56.338]; Breakthrough Project Grant

Conflict of interest

The authors declare no conflict of interest. Supplemental figure 3 DNA methylation of CT47 promoter region in pluripotent embryonic stem cells and differentiated embryonic bodies. High levels of DNA methylation was detected at CT47 promoter region in pluripotent and differentiated samples. One male (WA 14) and one female (WA 9) human embryonic stem cell and embryonic body pair was studied.

Supplemental figure legends