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Human Molecular Genetics Advance Access originally published online on June 29, 2006
Human Molecular Genetics 2006 15(15):2392-2399; doi:10.1093/hmg/ddl163
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The human X chromosome is enriched for germline genes expressed in premeiotic germ cells of both sexes

Michael Koslowski, Ugur Sahin{dagger}, Christoph Huber and Özlem Türeci*,{dagger}

Department of Internal Medicine III, Johannes Gutenberg-University, Obere Zahlbacher Street 63, 55131 Mainz, Germany

* To whom correspondence should be addressed. Tel: +49 61313933396; Fax: +49 61313933343; Email: tureci{at}uni-mainz.de

Received May 28, 2006; Accepted June 22, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The role of X-chromosomal genes in spermatogenesis has been subject to a number of studies in different organisms. Recently, it was proposed that the X chromosome has a predominant role in premeiotic stages of mammalian spermatogenesis. We analyzed the expression of a representative set of 17 X-linked and 48 autosomal germline-restricted genes in different stages of human germ cell development. In accordance with data from other species, we show that the human X chromosome is indeed significantly enriched for genes activated in premeiotic stages of spermatogenesis. In contrast to recent studies, however, we found that expression of these genes is not restricted to spermatogenesis, but is activated in oogenesis as well. Furthermore, we show that activation of this subset of genes merely depends on demethylation of their promoter regions. Moreover, our data suggest that genes activated in premeiotic stages of gametogenesis are sex-indifferent and are regulated by DNA methylation. Gene activation patterns involved in spermatocyte-specific differentiation, in contrast, appear to be initiated not before entry into meiosis and underlie a more complex regulation, presumably involving specific transcription factors and/or chromatin remodeling mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Sex chromosomes are subject to sex-specific selective evolutionary forces (1,2). Two hypotheses propose different outcomes for sex-linked genes that are preferentially expressed in one sex over the other. Rice's hypothesis (3) states that, because males carry a single X chromosome, any recessive allele arising on the X that gives males a reproductive advantage is immediately available for positive selection. Conversely, any negative effects of that allele in females are masked by the presence of a second X chromosome. Accordingly, it is assumed that male-specific genes are more likely to be present on the X chromosome. An alternative hypothesis claims that the X chromosome should be feminized because it is doubled in females when compared with males, providing evolution with more opportunity to act on genes benefiting females. According to recent data, these two hypotheses are not mutually exclusive. Although the latter is supported by global studies in Caenorhabditis elegans (4), Drosophila (5) and mice (6), indicating that germline male-biased genes are under-represented on the X chromosome and female-biased genes are enriched, recent observations imply that activation of germline genes at distinct stages of spermatogenesis needs to be factored in for a more refined analysis. In fact, in line with Rice's hypothesis, genes expressed during early spermatogenesis before the onset of meiotic sex chromosome inactivation have been reported to be enriched on the X chromosome of mice (6–9).

In this study, we analyzed 65 established germ cell-specific genes in different stages of human germ cell development. In accordance with previous reports obtained in mice, our data show that the human X chromosome indeed is enriched for genes activated during premeiotic stages of spermatogenesis. However, we demonstrate that these genes are not strictly specific for spermatogenesis, but are activated in oogenesis as well. Furthermore, our results suggest a distinctive role for DNA hypomethylation in the activation of X-chromosomal as compared to autosomal genes of the germ cell lineage.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIAL AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Selection of a representative sample of germline genes
To base our investigation of the role of the X chromosome in human spermatogenesis on a comprehensive and representative sample of genes, we revisited two independent sets of genes, which had been published as thoroughly characterized constituents of the germ cell-specific transcriptome and as such had been used in previously in similar meta-analytical studies. One of these data sets had been described by our group using a systematic data mining approach established for discovery of germline-specific genes (10,11). Importantly, we refer to genes that are selectively expressed in germ cells without any detectable expression in somatic tissues (Fig. 1). We combined this list with a second panel of gametogenic genes identified by subtractive hybridization studies by Wang et al. (7,9). We evaluated expression of all genes by specific end-point and quantitative real-time RT–PCR in a comprehensive panel of 21 human somatic tissue specimens encompassing the entire somatic body map (exemplified in Fig. 1) (further data in Koslowski et al. (11)]. Thereby, we determined 65 genes qualifying as authentically germ cell-specific (Table 1). Of these, 17 were X-linked and 48 were localized on autosomes. Notably, for the majority of the genes analyzed in the present study, an extensive literature survey yielded published experimental data including immunohistochemistry, in situ hybridization and RT–PCR proving restriction of expression to the germ cells and absence from the somatic compartment of testis (Supplementary Material, Table S1).


Figure 1631
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  		Figure 1. Expression analysis in gonadal and somatic tissues by (A) RT–PCR and (B) quantitative real-time RT–PCR exemplified for four genes analyzed showing specificity for gonadal tissues. TSGA10 and MTL5 are examples for genes originally described as germ cell-specific, yet showing robust expression in somatic tissues also. Those genes were discarded from our analysis (10,11).

 


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  		Table 1. The 65 germline genes analyzed sorted by chromosomal localization

 
Germline genes shared by both sexes are preferentially located on the X chromosome
First, we wanted to know whether genes accumulating on the X chromosome are specific for gametogenesis of one of the sexes. Because studies with pure populations of human gametogenic cells of all developmental stages are hampered by availability of specimen and technical feasibility, we resorted to bulk tissue specimens. Inclusion of testicular as well as ovarian tissues into this study allowed the discrimination of strictly spermatogenic genes from genes expressed in male as well as female gametogenesis. As proposed previously by Reinke et al. (4), we refer to the latter group of genes, which may have a common function in male and female germ cells, as germline-intrinsic.

Taking into consideration the paucity of germ cells in ovaries of adult females and the lack of germ cells in premeiotic stages, we only used specimen of adult ovarian tissue typed positive for the germ cell marker SYCP1 (12) and included fetal ovary as a source for premeiotic stages of oogenesis as well. Analysis of fetal and adult gonads from both sexes was performed by quantitative real-time RT–PCR (exemplified in Fig. 1B) and expression values were visualized in an expression heatmap (Fig. 2). We found that expression of 37 genes was strictly confined to spermatogenesis, whereas 28 genes could be assigned to the germline-intrinsic group (Fig. 3, Table 1). The chromosomal distribution of the 37 spermatogenesis-specific genes was significantly different from the germline-intrinsic group. The spermatogenesis-specific genes were distributed more homogenously throughout the genome with only four genes (11%) located on the X chromosome, whereas we observed a more skewed distribution for the germline-intrinsic genes with 13 genes (46%) accumulated on the X chromosome (P<0.001) (Fig. 3).


Figure 1632
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  		Figure 2. (A) Clustering of the 17 X-chromosomal and (B) 48 autosomal germline genes on the basis of transcript levels determined by quantitative real-time RT–PCR in fetal testis, fetal ovary, adult testis, PBMCs treated with 5-aza-dC (indicated as PBMC+) and untreated PBMCs (indicated as PBMC–). This led to the discrimination of genes restricted to spermatogenesis and genes expressed in both spermatogenesis and oogenesis as well (germline-intrinsic).

 


Figure 1633
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  		Figure 3. Chromosomal distributions of the analyzed 65 germline genes. Genes expressed in premeiotic stages of gametogenesis are depicted as circles; genes expressed during meiotic and postmeiotic stages are shown as squares. Filled symbols indicate inducibility by DNA demethylation; open symbols mean not inducible by DNA demethylation.

 
Genes activated early in gametogenesis are preferentially located on the X chromosome
In mice, it has been shown that only those spermatogenic genes expressed in mitotic and very early meiotic stages are preferentially located on the X chromosome, whereas genes expressed in the latter stages are depleted (6,7,9), indicating that meiotic sex chromosome inactivation is a driving force for the gene composition of the X chromosome in mammals. To analyze the gametogenic stage the genes are activated in, we exploited the differences in timing between male and female gametogenesis. Fetal ovary represents all stages of oogenesis from premeiosis to the prophase of meiosis I, which in adult ovary progress to the metaphase of meiosis I (13,14). As for spermatogenesis, fetal testis tissue represents premeiotic germ cells, whereas adult testis tissue harbours germ cells, most of which are past early meiosis (15). We deduced that genes belonging to different stages of germ cell development should have distinct but predictable expression patterns in fetal testis, fetal ovary and adult testis [for a more detailed explanation, see Koslowski et al. (11)]. We proofed this concept by testing the expression of genes, for which activation at defined gametogenic stages is well established (DMRT1 for premeiotic stages, synaptonemal complex proteins SYCP1 and SPO11 for meiotic stages and TNP2 and ODF1 for postmeiotic stages), in the three gonadal tissue types. We assigned all genes under investigation to the different stages of germ cell development by using the distinctive expression patterns in fetal testis, fetal ovary and adult testis (Fig. 2, Table 1). In line with previous studies in mice (6,7,9), we found genes expressed in premeiotic stages of germ cell development significantly enriched on the X chromosome (13/22, 59%) when compared with genes expressed in meiotic and postmeiotic stages (4/43, 9%; P<0.00005) (Fig. 3). In contrast to recent data, however, we observed that all premeiotically expressed genes with X linkage are not spermatogenesis-specific but are expressed in oogonia as well and thus are germline-intrinsic. Hence, in human spermatogenesis, as has been shown in mice (16), the majority of germ cell genes specific for spermatocyte differentiation seems not to be activated before entry into meiosis.

Expression of X-chromosomal germline genes is regulated by DNA methylation
These patterns suggested different mechanisms of transcriptional regulation. DNA methylation may contribute to gene silencing in cells (17) as demonstrated, in particular, for imprinted genes on autosomes (18–20), X-linked genes (21) and a subset of tissue-specific genes (21–24). It has been proposed that DNA demethylation could be the primary mechanism for the selective expression of tissue-specific genes with CpG-rich promoters, whereas those with CpG-poor promoters depend solely on the presence of tissue-specific transcription factors (22,25). To investigate the role of DNA demethylation in the control of gene expression, we tested inducibility of all genes in two independent assay systems. First, we treated non-expressing primary cells [peripheral blood mononuclear cells (PBMCs)] with the methylation-inhibiting drug 5-aza-2'-deoxycytidine (5-aza-dC). Secondly, we used an isogenic set of HCT116 human colorectal cancer cell lines in which the genes for the DNA methyltransferases DNMT1 and DNMT3b had been disrupted by targeted homologous recombination. This double-knockout variant has been reported to display a reduction in overall DNA methylation by greater than 95% (26). We observed that genes induced in one of these settings of genomic hypomethylation assays were also found to be activated in the other. Expression levels achieved by experimental DNA demethylation were comparable to those observed in physiologically expressing gonadal tissues (Figs 2 and 4A). Most interestingly, whereas expression of nearly all X-chromosomal genes (15/17, 88%), in particular those activated premeiotically, was easily induced by DNA demethylation, only a small fraction of the autosomal genes (9/48, 19%) could be activated that way (P<0.00001) (Fig. 3).


Figure 1634
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  		Figure 4. (A) 5-aza-dC treatment of PBMCs induces expression of genes to levels comparable with those observed in gonadal tissues. (B) Analysis of promoter methylation for six genes by bisulfite sequencing in untreated PBMCs and PBMCs treated with 5-aza-dC. Filled circles indicate methylated CpG and open circles indicate unmethylated CpG. (C) Relative methylation of promoters as analyzed by quantitative methylation-specific real-time PCR.

 
Notably, as reported earlier for tissue-specific genes that are regulated by DNA methylation (22), we identified CpG-islands in the promoter regions of the genes activated by DNA demethylation in our panel. For several of them, namely BAGE, GAGE, MAGE, XAGE1 and SPA17, bisulfite sequencing studies published by other groups had demonstrated that expression of these genes is regulated by the methylation state of their respective promoters (27–31). To further expand these data, we analyzed the methylation status of the promoter regions of five X-chromosomal genes (LUZP4, SAGE1, TAF7L, TKTL1, FTHL17) and one autosomal gene (TDRD1) that were induced in non-expressing PBMCs after treatment with 5-aza-dC by bisulfite sequencing (Fig. 4A). Although the promoters of all six genes were completely methylated in the untreated PBMCs, we found nearly complete demethylation of the promoters in PBMCs after 5-aza-dC treatment (Fig. 4B), indicating that expression of these genes indeed is regulated by methylation of their promoters. Methylation analysis by bisulfite sequencing of expressing germ cells in gonadal tissues is hampered by the presence of non-expressing somatic stromal tissue. Therefore, to analyze methylation in germ cells, we quantified the relative methylation of LUZP4, SAGE1, TAF7L, TKTL1, FTHL17 and TDRD1 promoters in gonadal tissues by methylation-specific real-time PCR. Oligos specific for the methylated promoter sequence were used for amplification of bisulfite-treated DNA from fetal and adult gonadal tissues. To confirm the specificity of the assay, we used bisulfite-treated DNA from untreated PBMCs and 5-aza-dC-treated PBMCs as controls. Consistent with the results of the bisulfite sequencing analysis, we found complete methylation of the promoter in untreated PBMCs and nearly complete demethylation in 5-aza-dC treated PBMCs for all six genes analyzed (Fig. 4C). For the germline-intrinsic genes LUZP4, SAGE1, TAF7L, TKTL1 and FTHL17, methylation levels in all gonadal tissues were distinctly reduced compared with untreated PBMCs. The remaining methylation of up to 40% most probably represents the non-expressing stromal cells of the gonads. Consistently, for TDRD1, the only spermatogenesis-specific gene analyzed, reduced methylation levels were detectable only in fetal and adult testis, whereas the non-expressing fetal ovary shows methylation levels equal to non-expressing untreated PBMCs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIAL AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Similar to previous investigations in other species (32), we have used transcription profiles as a measure for probing sex-biased function. One of the peculiarities of this study was that the selection of genes was based on accurate definitions and validity checking. We focused on germ cell-specific rather than germ cell-biased genes, which we authenticated by extensive expression profiling across somatic tissues. We also made a precise distinction between strictly spermatogenesis-specific and germline-intrinsic genes. Although this data set obviously does not comprise the entirety of spermatogenic and germline-intrinsic genes, we expect this sample to be representative.

In summary, our analysis disclosed highly significant diametric characteristics between X-chromosomal versus autosomal germ cell-specific genes with respect to their sex-specificity, the gametogenic stage they are activated at and the role of DNA demethylation for their transcriptional control (Fig. 5). Correlations between two of these features have been previously reported in model organisms. However, the multiparametric analysis we provide here elucidates previously unrecognized inter-relationships and provides novel insights.


Figure 1635
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  		Figure 5. Discrimination of X-chromosomal and autosomal genes according to sex-specificity, developmental stage of expression and inducibility by DNA demethylation. Fisher's exact test was used for statistical analysis.

 
The most surprising observation in this study is the prominent role of DNA demethylation in the activation of X-chromosomal but not autosomal genes of the germ cell lineage. The X chromosome has many features that are unique to the human genome. Particularly in gametogenic cells, which are the only cell lineage undergoing meiosis for haploidization, heterosomes take on a special role (33). This is exemplified by the specific cytological structure sex chromosomes form in meiotic spermatocytes, called the ‘XY’ body that is distinguished on the basis of its condensed chromatin structure involving alterations in DNA methylation and histone acetylation (34). Epigenetic regulation of differential gene expression which in general involves sophisticated structure-function interplay of DNA methylation, various histone modifications and chromatin remodeling involving a number of coding and non-coding transcripts seems to be methylation-biased on the X chromosome (35,36). DNA hypomethylation has been associated with epigenetic phenomena highly specific for the X chromosome, namely expression of the XIST gene (37) as well as of several gene clusters that escape X chromosome inactivation (38). However, previous data were based on case reports on single genes, whereas we have now shown that an entire set of temporally co-regulated X-linked genes depends solely on DNA demethylation for its activation. This relative independence of this X-chromosomal subset of germ cell-specific genes from specific transcription factors may also be the reason for their frequent ectopic activation in tumors (11,21).

It remains to be determined why germline-intrinsic genes that are expressed in early stages of gametogenesis accumulate on the human X chromosome. One would think that in early gametogenesis, which is dominated by mitotic proliferation and preparation for meiosis rather than sex-specific differentiation, generic transcriptional programs are required. This is supported by our finding that the majority of premeiotic genes in our representative sample turned out to be shared by both sexes. Most interestingly, in this stage of gametogenesis, germ cells are characterized by global DNA demethylation in preparation for sex-specific de novo methylation (33,39).

This may provide a simple mechanism of co-regulation, as genes which depend on promoter hypomethylation alone for transcription initiation and which are embedded into an appropriate epigenetic environment would be activated in a temporally aligned manner by default. The X chromosome may provide such an environment for DNA methylation to act as a prominent transcriptional regulator and thus may be the most suitable localization for genes involved in these early transcriptional programs. Starting with or after entry into meiosis, transcriptional programs aimed at sex-specific differentiation have to come to the front. As shown in this study, genes activated after meiosis are indeed male-specific and are located on autosomes. Sex-specification seems to underlie a more complex regulation, presumably involving specific transcription factors and/or regional chromatin remodeling mechanisms (40,41).


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Tissues and cell lines
Human tissues were obtained in an anonymous fashion from the tumor tissue bank of the program project (SFB432) supported by the Deutsche Forschungsgemeinschaft, derived from surplus tissue from the routine surgical pathology service. Fetal testis (24-week gestation) and fetal ovary (18- and 20-week gestation) from abortions were stored at –80°C until use. To induce DNA demethylation, PHA-activated PBMCs were cultured in RPMI 1640/10% FCS supplemented with 2 µM or 10 µM 5-aza-2'-deoxycytidine (Sigma) for 72 h. HCT116 cell lines knocked out for DNMT1 or DNMT3b as well as the double-knockout cell lines were kindly provided by Bert Vogelstein and cultured in McCoy's 5A/10% FCS.

RNA-isolation and end-point and real-time RT–PCR
Total cellular RNA was extracted from frozen tissue specimens using RNAeasy Mini Kit (Qiagen), primed with a dT18 oligonucleotide for RT–PCR or random hexamers for quantitative real-time RT–PCR, and reverse-transcribed with Superscript II (Invitrogen) according to manufacturer's instructions. Integrity of the obtained cDNA was tested by amplification of p53 transcripts in a 30-cycle PCR (sense: 5'-CGT GAG CGC TTC GAG ATG TCC G-3'; antisense: 5'-CCT AAC CAG CTG CCC AAC TGT AG-3'; annealing temperature 67°C). To authenticate germ cell-specificity of genes, tissue distribution was assessed by RT–PCR. Expression analysis was performed in a comprehensive set of normal tissues (spleen, thymus, mammary gland, liver, ovary, prostate, lymph node, uterus, kidney, thyroid, small intestine, colon, pancreas, adrenal gland, esophagus, lung, skin, resting and activated PBMCs, brain and testis). Primers spanning exon/intron boundaries sequences and annealing temperatures are provided in Supplementary Material, Table S2. For end-point RT–PCR analysis of individual gene transcripts, 0.5 µl first-strand cDNA was amplified with transcript-specific oligonucleotides (Invitrogen) using 1 U HotStarTaq DNA polymerase (Qiagen) in a 30 µl reaction according to manufacturer's instructions. In each experiment, a template-free negative control and testis as positive control were included. Quantitative real-time RT–PCR analysis was performed using the ABI PRISM 7300 Sequence Detection System instrument and software (Applied Biosystems) with QuantiTect SYBR Green PCR Kit (Qiagen). Reactions were performed for 40 cycles in triplicates with specific primers (300 nM each) with initial denaturation/activation for 15 min at 95°C, 30 s denaturation at 94°C, 30 s annealing and 30 s amplification at 72°C. The relative expression level of specific transcripts was calculated with respect to the internal standard 18sRNA (sense: 5'-CGA TGC TCT TAG CTG AGT GTC-3'; antisense: 5'-TAA CCA GAC AAA TCG CTC CAC-3'; 65°C) to normalize for variances in the quality of RNA and the amount of input cDNA. Expression levels were normalized to non-expressing PBMCs using {Delta}{Delta}CT calculation. In each experiment, a template-free negative control and testis as positive control were included.

DNA extraction and bisulfite sequencing
DNA from cells was extracted using DNAeasy Tissue Kit (Qiagen) and subjected to bisulfite modification using the CpGenome DNA Modification Kit (Intergen) according to manufacturer's instructions. In brief, 1 µg of genomic DNA was denatured by sodium hydroxide and then chemically modified by sodium bisulfite treatment for 20 h. This converts the unmethylated cytosine to uracil, whereas methylated cytosine remains unchanged. The modified DNA was recovered by ethanol precipitation and resuspended in PCR-grade water. For mapping of methylated cytosine residues, reaction products were amplified with primers specific for modified promoter sequences not covering any CpG dinucleotide. The primer sequences were LUZP4 (sense1: 5'-GTA GTT AAT TAT ATA GTG TTT TAG GTG GAG AG-3'; antisense1: 5'-TAA ACT AAC CTA ATC CTC CAA TCA TAA CCT C-3'; sense2: 5'-GAG GTT ATG ATT GGA GGA TTA GGT TAG-3'; antisense2: 5'-CTC AAA ACC AAT AAA CCC AAA C-3'; sense3: 5'-GTT TGG GTT TAT TGG TTT TGA G-3'; antisense3: 5'-CAA CCT CCC TCA ATT CCC CAC C-3'), SAGE1 (sense1: 5'-GTA GTA TTT TAT AAA ATG GTA GTG AAG TTG TGG-3'; antisense1: 5'-ATC AAC TTC CCT TCC TTC CAT C-3'; sense2: 5'-GAT GGA AGG AAG GGA AGT TGA T'; antisense2: 5'-AAA ATT CTA TAT TCC TCT ACC TCT ACC-3'), TAF7L (sense1: 5'-GTT TTT AAG AAT ATT GTA TAG TAA ATG TAA AAT TGG-3'; antisense1: 5'-TCT CCA CTA CCT AAC ACT CAC CCA AAC-3'; sense2: 5'-GTT TGG GTG AGT GTT AGG TAG TGG AG-3'; antisense2: 5'-CTA CTT TCA TAA ATA ATT TCA AAC CC-3'; sense3: 5'-GGG TTT GAA ATT ATT TAT GAA AGT AG-3'; antisense3: 5'-CCA CCT ACC TTT CCC TTC C-3'), TKTL1 (sense1: 5'-GTT ATA GTT TGG GTT GTT GTG GGG GAG G-3'; antisense1: 5'-CCA TTC TCA TCT TCT CTC CTA CAA TCT C-3'; sense2: 5'-GAG ATT GTA GGA GAG AAG ATG AGA ATG G-3'; antisense2: 5'-CCC CTA TCA AAT CTA ACC TCC TC-3'), FTHL17 (sense1: 5'-GTT GTT TGA TAT AAG TTT TGT TTT GTT GTT TAG GTT GG-3'; antisense1: 5'-CAA AAC ATA AAA ACC AAC CTA ACC AAC-3'; sense2: 5'-GTT GGT TAG GTT GGT TTT TAT GTT TTG-3'; antisense2: 5'-AAA ACA ACA ACT AAA TAA CAA AAA TAA C-3'; sense3: 5'-GTT ATT TTT GTT ATT TAG TTG TTG-3'; antisense3: 5'-CAA ATT CTA CAA CCT CAT CAA CTT C-3') and TDRD1 (sense1: 5'-GTG TTT ATT TTT TAA GAT TAG GTA GAG GTT G-3'; antisense1: 5'-CCT TCA TAC AAA CCC TCT CCC TCC-3'; sense2: 5'-GGA GGG AGA GGG TTT GTA TGA AGG-3'; antisense2: 5'-CAC TAA ATC CAC ATA CAA ATT TCT C-3'). Amplification products were cloned into TA cloning vector (Invitrogen) and 10 individual clones per promoter were sequenced.

Quantitative methylation-specific PCR
After sodium bisulfite conversion, the methylation analysis was performed by fluorescence-based, real-time PCR assays. For each gene, primers were designed to specifically amplify bisulfite-converted DNA of the methylated, but not hypomethylated promoter. The primer sequences were LUZP4 (sense: 5'-TGA TAG GAG ATT TGG TGA GGG GTA AC-3'; antisense: 5'-CGC CAC ACA CTC CGA AAT AAA ACG CG-3'), SAGE1 (sense: 5'-TGG GAG TGA TGT TTA TGG GGA GGC-3'; antisense: 5'-CCT AAT TCT CCC GCT AAC GCC AAC G-3'), TAF7L (sense: 5'-GTT TGG GTG AGT GTT AGG TAG TGG AGA C-3', antisense: 5'-CCT CGA AAC ACT CCA TAA CCC ACC TCG-3'), TKTL1 (sense: 5'-GGA GCG GTA GGT GGA GGG AGT GGC AC-3', antisense: 5'-ATC TAA AAA CCC ACT CCT ACG TCT CCG ACG-3'), FTHL17 (sense: 5'-GGT CGG GAT TTT CGA CGG AAG TTA GC-3', antisense: and 5'-CAC ACG AAC GAA ATC GAT ACG ATA ACG-3'), TDRD1 (sense: 5'-GTT GTT TTC GGG GAA GGC GGA GGG AAT AC-3', antisense: 5'-CGC CTA AAA TAC GCC CTA CAC TTC CCT CG-3'). Amplification of ß-actin (ACTB) with primers (sense: 5'-TGG TGA TGG AGG AGG TTT AGT AAG T-3', antisense: 5'-AAC CAA TAA AAC CTA CTC CTC CCT TAA-3') designed to amplify independent of the ACTB methylation status was used to normalize for input DNA. Specificity of the reactions for methylated DNA was confirmed using bisulfite-modified DNA from PBMCs, either treated with 5-aza-dC or untreated, as controls. The percentage of methylated alleles at a specific locus was calculated by dividing the gene/ACTB ratio of a sample by the gene/ACTB ratio of bisulfite-modified Universal Methylated DNA (Chemicon) and multiplying by 100. Quantitative real-time PCR analysis was performed using the ABI PRISM 7300 Sequence Detection System instrument and software (Applied Biosystems) with QuantiTect SYBR Green PCR Kit (Qiagen). Reactions were performed for 40 cycles in triplicates with specific primers (300 nM each) with initial denaturation/activation for 15 min at 95°C, 30 s denaturation at 94°C, 30 s annealing and 30 amplification seconds at 72°C.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 432 and grant TU 115/2-1), the European Commission (EUCIP) and the MAIFOR Program of the Johannes Gutenberg-University.

Conflict of Interest statement. None of the authors have any direct conflicts of interest with the work presented here.


   FOOTNOTES
 
{dagger} Both the authors contributed equally. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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