RevSex duplication-induced and sex-related differences in the SOX9 regulatory region chromatin landscape in human fibroblasts

Abstract

It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of "chromatin-CGH") and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14-15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.

Keywords: 46,XX DSD; ChIP-on-chip; RevSex duplication; SOX9; TESCO; chromatin; epigenetics; sex determination.

Figure 1. An UCSC genome browser panel with custom tracks showing the differences in H3K4me3 (top), H3K27me3 (middle) and H3K9me3 (bottom) chromatin profiles between the 46,XX ovotesticular DSD male and his father in the area containing the RevSex duplication (marked with a black line). The curve displays the ChIP-on-chip signal of the index case after subtraction of the signal of his XY-father. The red lines mark the position of predicted SRY and SOX9 binding sites (“HMR Conserved Transcription Factor Binding Sites”), all with high Z-scores for SRY binding: 3.29 for SRY-1 and 3.13 for SRY-2. SRY-2 had in addition a high degree of genomic sequence conservation beyond the human/mouse/rat comparison that the Z-score calculations are based on; See the UCSC browser “Vertebrate Multiz Alignment & Conservation (46 Species)” track for details. A position of difference commented in the text is marked with “A.”

Figure 2. An UCSC genome browser panel with custom tracks displaying the differences in H3K4me3 (top), H3K27me3 (middle) and H3K9me3 (bottom) chromatin profiles between the XX-DSD male and his XY-father in a small region 158 kb downstream of the RevSex duplication containing a transcription factor/CTCF binding sites (UCSC genome browser track “HMR Conserved Transcription Factor Binding Sites”) called DMR2 in Table 1 and marked B in the figure. The upstream binding site (red line) corresponds to a DNaseI hypersensitivity site and is also predicted to bind SRY and SOX9 (corresponds to the SRY-2/SOX9 site in Fig. 1). The downstream binding site (green line) contains a CTCF binding site (ENCODE immunoprecipitation data) and a putative SRY binding site 3 kb downstream. It is important to note that SRY and SOX9 binding sites are computationally predicted based on human, mouse and rat conservation of binding sites as described in the Transfac database and have not been validated experimentally (see UCSC genome browser table “HMR Conserved Transcription Factor Binding Sites” description for more details).

Figure 3. A UCSC genome browser panel with custom tracks displaying the signal-to-input chromatin profiles (H3K4me3, H3K27me3, and H3K9me3) of the XX-DSD case (upper profiles) compared with his RevSex duplication-carrier father (lower profiles) in the area from SOX9 and 120 kb upstream (70 000–70 126 kb from 17pter, hg19). The red line called TESCO corresponds to the position of the TESCO-ECR (70 103 197–70 103 373 bp from 17pter, hg19), the green line marked C to DMR3 in Table 1, and the dashed line the start of the SOX9 gene. The same position marks and more detailed explanations can be found in Figure 5.

Figure 4. An UCSC genome browser panel with custom tracks showing the average signal-to-input chromatin profiles (H3K4me3, H3K27me3, and H3K9me3) of control females (upper profiles, n = 3) and control males (lower profiles, n = 3) in the 736 kb region from 69,390,000 to 70,126,000 (hg19) after 1 kb binning of data. A, C, and D mark positions of differences commented in the text. The dashed line marks the start of the SOX9 gene.

Figure 5. An UCSC genome browser panel with custom tracks displaying the difference between control male (n = 3) and control female (n = 3) chromatin profiles of the part SOX9 locus from the gene itself and 120 kb upstream (70,000 – 70,126 kb from 17pter, hg19). The profiles shown are H3K4me3 (top), H3K27me3 (middle), and H3K9me3 (bottom). The red line called TESCO marks the position of the TESCO-ECR (70,103,197–70,103,373 bp from 17pter, hg19) and the green line the position of DMR3 in Table 1. The predicted SRY-3/SOX9 binding site had a Z-score of 3.45 for SOX9 and 2.41 for SRY, while the TESCO enhancer had a Z-score of 2.58 for SOX9 and 2.66 for SRY (UCSC, “HMR Conserved Transcription Factor Binding Sites”). In addition, the TESCO enhancer had a high degree of genomic sequence conservation beyond the human/mouse/rat comparison used to calculate the Z-score (UCSC, “Vertebrate Multiz Alignment & Conservation (46 Species)”). The TESCO SRY binding site has been experimentally verified.

Figure 6. A schematic drawing of our major findings: In females the RevSex duplication (black line, region A) has more repressive chromatin with a more “active” putative enhancer element (orange dot) than in males. In the XX-DSD case similar findings are done with the exception of a more “open” SOX9 enhancer/promoter (region C). In males a downstream CTCF binding site is enriched in H3K27me3 (PRC2-related) chromatin compared with females (region B), and the SOX9 enhancer/promoter region (region C) is more open (red dot). This region is even more open in the XY RevSex carrier (orange dot). On the right side a comparison of the DMR3 H3K4me3 peaks (region C) can be seen with internal comparisons to the H3K4me3 peaks of the SOX9 promoter with peak ratios shown on the right. Chromatin color codes are follows: black, H3K9me3; blue, H3K27me3; red, H3K4me3; orange, stronger H3K4me3.