Post by Admin on Nov 23, 2023 20:09:50 GMT
Fig. 4 Enh13 is bound by SRY and SOX9 in vivo.
(A) A schematic representation of the locations of the primers used for ChIP experiments in Enh13. BS, binding site. (B) ChIP-qPCR assay of E11.5 mouse genital ridges after immunoprecipitation with anti-cMYC antibody. Data are presented as the level of enrichment of SRY-MYC–positive relative to SRY-MYC–negative genital ridges, meaning that values greater than 1 (marked by the dotted line) represent specific enrichment. The primers used span the putative SRY binding site in Enh13 and TESCO [around the SRY R6 site; see (16)] and a negative control region on chr11. Data are the means ± SD (n = 2); *P ≤ 0.05, **P ≤ 0.01, ****P < 0.0005 (Student t test). (C) ChIP-qPCR assay of E13.5 mouse testes after immunoprecipitation with anti-SOX9 antibody. The primers used span the putative SOX9 binding site in Enh13 and TESCO [around the SOX9 R1 site; see (16)] and a negative control region on chr11. Data are the means ± SD (n = 3). ****P < 0.0005; ns, not significant (Student t test). Unr ab, unrelated antibody (rabbit IgG isotype) used as a control. (D) ChIP-seq with the use of anti-SOX9 antibody and E90 fetal bovine testis. The bovine (bov) Enh13 is indicated by the gray box (at cow Bostau8 chr19: bp 60,063,628 to 60,064,165). The asterisk denotes the peaks with a false discovery rate of <0.05 in the two bovine datasets. y-axis numbers represent counts. The input tracks represent sequencing reads of chromatin input. The bovine Sox9 gene is indicated by the arrow and is 570 kb downstream (to the left) of Enh13.
ChIP assays revealed SOX9 to be bound at similar levels to both Enh13 and TESCO in cells from E13.5 testes (Fig. 4C). In addition, SOX9 ChIP-seq data obtained with bovine embryonic testis (25) revealed a strong peak localizing to the conserved syntenic region of Enh13 (537 bp long, 570 kb 5′) (Fig. 4D). This suggests that, like TES, Enh13 is used by SOX9 to autoregulate SOX9 expression and that this interaction is conserved in mammals. Unlike several other gonadal enhancers, Enh13 appears to be well conserved, has a clear role in mice to initiate up-regulation of Sox9 expression in response to SRY activity, and may contribute to maintaining Sox9 expression. This makes it very likely to play a similar role in humans, given its location within the XY SR region (20, 26). If it does play a similar role, heterozygosity for Enh13 deletions in humans should mimic heterozygosity for null mutations in SOX9, with XY female sex reversal occurring in about 70% of cases but perhaps without other CD phenotypes.
It is clear from our data and those of others that the upstream regulatory region of Sox9 is very complex. Our screens with gonadal cells revealed 33 potential enhancers distributed over 1.5 Mb. Transgenic assays used to test the most promising 16 enhancers revealed 4 that produced expression in the gonads, whereas the majority did not. This “hit rate” of 25% agrees with the results of some other studies (19) and merits caution in interpreting data based solely on the accessibility of chromatin and histone marks. However, several of these putative enhancers are bona fide enhancers in other locations; for example, Enh29, mapping about 70 kb 5′, is equivalent to SOM, an enhancer active in many tissues, excluding the gonads (27). Others may have distinct roles; Enh11 contains a putative CTCF binding site. In addition, several putative enhancers, notably Enh4, -5, -8, and -9, appear to be open in both granulosa and Sertoli cells, with Enh8 even more so in the former. These may contribute to the low level of Sox9 expression seen in supporting cell precursors, but they may also represent sequences required to repress Sox9, which might not be detected by transgenic reporter assays.
The notion of redundancy or “shadow enhancers” within a regulatory region is well established (28, 29), and recent data suggest that deletion of single, even “ultraconserved” enhancers from developmentally important genes can have at most subtle if not undetectable effects (30, 31). It is therefore remarkable to see that deleting Enh13 alone phenocopies the loss of Sox9 itself within the supporting cell lineage (9, 32). Substantial evidence points to the time-dependent action of SRY on Sox9. If Sox9 fails to reach a critical threshold within a few hours, then ovary-determining and/or antitestis factors, such as Wnt signaling, accumulate to a sufficient level to repress Sox9 and make it refractory to male-promoting factors, including SRY, even though expression of the latter persists in XY gonads when Sertoli cells fail to differentiate (33). We suggest that Enh13 is an early-acting enhancer, such that without it Sox9 transcription fails to increase to a level where the other enhancers can act before the gene is silenced. It is only later that TES, and perhaps other enhancers such as Enh14 and Enh32, begins to act in a more redundant fashion, although it is conceivable that each enhancer has a major role to play during distinct phases of Sertoli cell development from the fetal to the adult testis. It will be of interest to determine how Enh13 activity cascades into the recruitment of the other enhancers.