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Post by Admin on Nov 21, 2023 20:05:15 GMT
Scientists have turned male mice into females by snipping out strands of their DNA in work that could shed light on sexual development disorders which arise in humans. The male mice grew ovaries and female genitalia instead of the more conventional male anatomy after researchers removed small chunks of DNA from the animals’ genetic code. The findings could help explain why people with XY chromosomes who are missing similar strands of DNA can go on to develop female sex organs, the scientists say. Males are typically born with X and Y chromosomes, while females have two Xs. Researchers at the Francis Crick Institute in London showed that they could reverse the sex of male mice by deleting a chunk of DNA called enhancer 13, or Enh13 for short. Like 98% of the genome, this section of DNA does not carry any genes that are used to make proteins, the crucial building blocks of living organisms. “For the first time we’ve demonstrated sex reversal after changing a non-coding region of DNA,” said Robin Lovell-Badge, a geneticist who led the research at the Francis Crick Institute. “We think Enh13 is probably relevant to human disorders of sex development and could potentially be used to help diagnose some of these cases.” At least half of the sexual development disorders seen in humans have unexplained genetic causes, the scientists say. “The analysis of such patients has mostly focused on the parts of genes that encode proteins, ignoring the parts that control the activity of the gene.” Lovell-Badge said. Mammal embryos are destined to grow ovaries and become females unless the sex organs get enough of a protein called SOX9 early on in the womb. The protein turns the organs into testes which then steer the embryo down the path to maleness. In the earliest stages of development, levels of SOX9 are driven by a gene on the Y chromosome, explaining why males typically develop testes. Writing in the journal, Science, the researchers show that Enh13 boosts SOX9 levels at precisely the right time to produce testes. When it was snipped out of the genetic code, male mice went on to become females. “Our study highlights the important role of what some still refer to as ‘junk’ DNA, which makes up 98% of our genome,” said Nitzan Gonen, another geneticist at the Francis Crick Institute, and first author on the study. “If a single enhancer can have this impact on sex determination, other non-coding regions might have similarly drastic effects.” Lovell-Badge added: “Even if there is no immediate solution, this can help patients come to terms with their condition and it helps clinicians decide on the best way to manage it. However, it is the understanding of how genes are regulated that might ultimately have the greatest benefit. As we learn more we may be able to develop ways to modulate gene activity to solve a wide range of clinical problems and not just those affecting testis or ovary development.” www.science.org/doi/10.1126/science.aas9408 |
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Post by Admin on Nov 21, 2023 20:09:30 GMT
Sex reversal following deletion of a single distal enhancer of Sox9 Sox9 regulation during sex determination Sex determination is regulated by the Sox9 gene. During testis differentiation, this gene is directly targeted by the product of the Y chromosome–encoded gene Sry. The regulatory region of Sox9 is complex, which is typical of genes with multiple roles in development. Gonen et al. find that a single far-upstream 557–base pair element is critical for up-regulating Sox9. Without it, XY mice develop as females instead of males. The 557–base pair enhancer is conserved, likely to be relevant to human disorders of sex differentiation, and probably essential because it acts early in a time-critical process, and any failure allows ovary-specific factors to dominate. Science, this issue p. 1469 Abstract Cell fate decisions require appropriate regulation of key genes. Sox9, a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Sox9. Although others are redundant, enhancer 13 (Enh13), a 557–base pair element located 565 kilobases 5′ from the transcriptional start site, is essential to initiate mouse testis development; its deletion results in XY females with Sox9 transcript levels equivalent to those in XX gonads. Our data are consistent with the time-sensitive activity of SRY and indicate a strict order of enhancer usage. Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in humans is associated with XY sex reversal, suggesting that it is also critical in humans. The regulation of genes with important roles in embryonic development can be complex, involving multiple, often redundant enhancers, silencers, and insulators (1, 2). The genes may have a poised epigenetic state prior to their expression, and their activation or repression may involve positive or negative feed-forward loops. This complexity is likely to be amplified when the gene has functions in more than one tissue, given that the regulatory elements required for each are often interspersed and necessitate dynamic alterations in chromatin conformation (1, 2). The developing gonads constitute an interesting system in which to explore questions of gene regulation during development (3). Most of the cell lineages are bipotential, with the ability to give rise to cell types typical of either ovaries or testes, and many genes that become associated with male or female fate begin by being expressed at equivalent, although usually low, levels in supporting cell precursors of both XX and XY gonads (4–6). In mammals, the Sry gene encodes a protein that is transiently expressed and initiates testis and subsequent male development by triggering cells of the supporting cell lineage to differentiate into Sertoli cells rather than granulosa cells typical of ovaries (7). Sox9, the main target of SRY, is critical for the differentiation of Sertoli cells and then functions along with other transcription factors, notably Sox8 and then Dmrt1, for Sertoli cell maintenance (4–6). Both gain- and loss-of-function studies in mice and humans demonstrate that Sox9 plays a key role in testis determination (8–13). Notably, humans heterozygous for null mutations develop campomelic dysplasia (CD) [Online Mendelian Inheritance in Man (OMIM) entry 114290] (11), a severe syndrome where 70% of XY patients show female development (12, 13). Sox9 functions in many embryonic and adult cell types (14), and genetic and molecular evidence suggests that its regulatory region is spread over a gene desert of at least 2 Mb 5′ to the coding sequence (15). The only enhancer known to be relevant for expression in Sertoli cells was TES, a 3.2-kb element mapping 13 kb 5′ from the transcriptional start site, and its 1.4-kb core, TESCO (16). Targeted deletion of TES or TESCO reduced Sox9 expression levels in the early and postnatal mouse testis to about 45% of normal but did not result in XY female development (17). We therefore used several unbiased approaches to systematically screen for additional gonad enhancers upstream of mouse Sox9. We used deoxyribonuclease I hypersensitive site sequencing (DNaseI-seq) data obtained with embryonic day 13.5 (E13.5) and E15.5 sorted Sertoli cells (18). From 33 putative enhancers, we chose only those positive at both stages (14 enhancers) for in vivo validation by transgenic assays (Fig. 1A and fig. S1). In parallel, we carried out ATAC-seq (assay for transposase-accessible chromatin using sequencing) on XY and XX gonads, which permitted the use of fewer sorted cells at E10.5, an early bipotential stage, and E13.5, when gonadal sex is already determined (figs. S1 and S2 and methods). Most putative enhancers discovered by DNaseI-seq were evident in the E13.5 XY ATAC-seq data; however, we used this assay to include two more putative enhancers in the in vivo screen: enhancer 1 (Enh1) and Enh14 (Fig. 1A and fig. S1). Chromatin immunoprecipitation sequencing (ChIP-seq) was also performed for H3K27ac, a histone modification that marks active enhancers (fig. S1). www.science.org/doi/10.1126/science.aas9408 |
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Post by Admin on Nov 21, 2023 20:17:50 GMT
 Fig. 1 Enh13 is a testis-positive enhancer of Sox9 located within the XY SR region. (A) A schematic representation of the gene desert upstream of the mouse Sox9 gene and the locations of the putative enhancers identified by ATAC-seq and DNaseI-seq that were screened in vivo with transgenic reporter mice. Enhancers that did not drive gonad expression of LacZ are shown in gray. Enhancers that drove testis-specific and ovary-specific LacZ expression are shown in blue and pink, respectively. The mouse regions that show conserved synteny with the human XY SR and REV SEX are depicted in green and purple boxes, respectively. (B) Enh13 (gray box) is located at the 5′ side of the 25.7-kb mouse equivalent XY SR locus (heavy black line). Data from DNaseI-seq (black) on E15.5 and E13.5 XY sorted Sertoli cells and ATAC-seq on E13.5 sorted Sertoli cells (blue) and granulosa cells (purple), as well as E10.5 sorted somatic cells, at the Enh13 genomic region are presented. Peaks correspond to nucleosome-depleted regions and are marked by a black horizontal line if they are significantly enriched compared to flanking regions, as determined by model-based analysis of ChIP-seq, and present in at least two biological replicates. The gray box overlaying the peak indicates the cloned fragment. Green areas represent sequence conservation among mice, humans (Hu), rhesus monkeys (Rh), cows (Co), and chickens (Ch) (sequence conservation tracks were obtained from the University of California–  Cruz). (C) β-Gal staining (blue) of E13.5 testes and ovaries from two representative independent stable Enh13 transgenic (Tg) lines. Scale bars, 100 μm. All 16 putative enhancers were cloned upstream of an Hsp68 minimal promoter and the reporter gene LacZ and used to generate transgenic mice (2, 19) (table S1). For initial screens, we performed transient analyses at E13.5. Twelve enhancers failed to produce any gonadal β-galactosidase (β-Gal) activity, although many showed staining in other tissues in which Sox9 is normally expressed, such as chondrocytes, brain, and spinal cord (fig. S3). The remaining four showed gonad expression, and these constructs were reinjected to generate stable lines in order to better study their activity in both males and females during development. Enh8 [672 base pairs (bp) long, 838 kb 5′] conferred robust β-Gal activity in the ovary, whereas it was barely present in the testis at E13.5 (Fig. 1A and fig. S4B). This may be due to Enh8 being taken out of its original genomic context; notably, ATAC-seq revealed a much stronger peak in granulosa cells than in Sertoli cells (fig. S4A). In contrast, Enh14 (1287 bp long, 437 kb 5′) showed robust testis-specific β-Gal activity (Fig. 1A and fig. S5B). DNaseI-seq, ATAC-seq, and H3K27ac ChIP-seq data all suggest that this enhancer is active and open only in Sertoli cells (figs. S1 and S5A). To test this candidate, we used genome editing to delete Enh14. However, Enh14 deletion did not alter expression of Sox9; its target gene Amh; or Foxl2, a marker of granulosa cells, in E13.5 XY gonads (fig. S5D), indicating that Enh14 has a redundant role, at least in the embryo. Enh32 (970 bp long, 10 kb 5′) is also testis specific but very weak and restricted to a domain close to the mesonephros (Fig. 1A and fig. S6, B and C). ATAC-seq, DNaseI-seq, and H3K27ac ChIP-seq data suggest that Enh32 is a Sertoli cell enhancer, although weak peaks were seen in the granulosa cell samples (figs. S1 and S6A). The remaining enhancer, Enh13 (557 bp long, 565 kb 5′), is highly conserved among mammals and is located toward the distal 5′ end of a 25.7-kb region in mice that shows conserved synteny with a 32.5-kb region upstream of human SOX9 termed XY SR, the deletion of which is associated with sex reversal (20) (Fig. 1, A and B, and fig. S1). Enh13 shows the strongest Sertoli cell–specific peak within this region in both the DNaseI-seq and ATAC-seq data. H3K27ac ChIP-seq data mark Enh13 as active in both Sertoli and granulosa cells, which may support the observation that some transgenic lines also exhibit β-Gal activity in the ovary (Fig. 1C and figs. S1 and S7A). β-Gal expression is clearly within Sertoli and granulosa cells (fig. S7C). ATAC-seq data from E10.5 genital ridges show that Enh13 is not open at this stage, irrespective of chromosomal sex (Fig. 1B and fig. S1), suggesting that it opens coincident with specification of the supporting cell lineage from SF1-positive cells of the coelomic epithelium (5). Genome editing was used to derive mice homozygous for deletions of Enh13. Homozygous deletion always led to XY female development, whether in a TES mutant background or in a wild-type background (Fig. 2 and figs. S8 to S10). The latter result was surprising, because if TES accounts for 55% of Sox9 expression in early Sertoli cells, any additional enhancer(s) should not account for more than 45% and, when this enhancer is deleted, levels of Sox9 should remain higher than the threshold of ~25% below which sex reversal might be expected (17). Nevertheless, whereas XY Enh13+/− embryos still undergo normal testis development, XY Enh13−/− embryos produce ovaries indistinguishable from those of XX wild-type embryos, with no signs of testis cords or a coelomic vessel (Fig. 2, B and C, and fig. S10). Immunofluorescence analysis of E13.5 and 6-week-old XY Enh13+/− and Enh13−/− gonads for SOX9 and FOXL2 showed that the former are still testes whereas the latter are fully sex-reversed ovaries (Fig. 2C and fig. S10D). Similar analysis of XX gonads with Enh13 deletion did not show any obvious phenotype (fig. S11). |
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Post by Admin on Nov 21, 2023 20:28:51 GMT
 Fig. 2 Deletion of Enh13 leads to complete XY male-to-female sex reversal. (A) A schematic representation of the locations of Enh13 and TES upstream of Sox9. Blue and purple arrows represent the external and internal single guide RNAs, respectively, used to delete Enh13. Black arrows represent the PCR primers used to genotype embryos and mice with Enh13 deletion. Chr11, chromosome 11. (B) Bright-field (BF) pictures and hematoxylin and eosin (H&E)–stained sections of E13.5 XY Enh13+/+, Enh13+/−, and Enh13−/− and XX Enh13+/+ gonads. (C) Immunostaining of E13.5 XY wild-type, Enh13+/−, and Enh13−/− and XX wild-type gonads. Gonads were stained for Sertoli marker SOX9 (green), granulosa marker FOXL2 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Sex-reversed gonads are indistinguishable from wild-type XX gonads, whereas the heterozygous deletion does not appear to alter testis morphogenesis. Scale bars in (B) and (C), 100 μm. The Enh13 deletion was generated in a C57BL/6J genetic background, which is sensitized toward XY female sex reversal (21). To test the strength of the deleted allele, we therefore backcrossed the deletion into a mixed C57BL/6J × CBA background. As before, XY heterozygotes presented as normal fertile males whereas homozygotes showed full male-to-female sex reversal (fig. S12). We could detect no difference in gonadal phenotypes between Enh13−/−:TES+/+ and Enh13−/−:TES−/− embryos or mice, suggesting that homozygosity for the Enh13 deletion alone reduced Sox9 levels well below the critical threshold required for testis development, which had been determined at E13.5 (17) (figs. S8 and S10). However, examining levels of gene expression at this stage when there is sex reversal will be uninformative because factors such as WNT4 and FOXL2 repress Sox9 once ovary development begins (5). We therefore analyzed Sox9, Sry, Sf1, and Foxl2 mRNAs at E11.5, during the brief period when gonadal sex is being determined. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed that XY Enh13+/− and Enh13−/− genital ridges expressed 58 and 21% of the wild-type levels of Sox9 mRNA, whereas XY TES+/− and TES−/− genital ridges showed 55 and 50%, respectively (Fig. 3A). Control XX genital ridges contained 18% of the Sox9 mRNA levels found in XY genital ridges (Fig. 3A). Therefore, E11.5 XY Enh13−/− gonads express Sox9 at levels close to those of XX gonads at the same stage, explaining the observed complete sex reversal. Deleting one or two copies of TES had relatively little effect at E11.5, especially compared with the effect at E13.5, in contrast to the results with Enh13 deletions at E11.5 (Fig. 3A) (17). This again supports the conclusion that Enh13 plays a more substantial role than TES during early gonadal development. |
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Post by Admin on Nov 22, 2023 19:40:26 GMT
 Fig. 3 Enh13 regulates the expression of the Sox9 gene in vivo. (A to D) RT-qPCR analysis of genes involved in male (Sox9, Sry, and Sf1) and female (Foxl2 and Sf1) gonadal sex determination in E11.5 XY gonads with Enh13 deletion and/or TES deletion (18 tail somites). Data are presented as mean 2−ΔΔCt values normalized to the expression of the housekeeping gene Hprt. The sample sizes (n) listed below each genotype are the numbers of individuals. Error bars show SEM of 2−ΔΔCt values. P values are represented above the relevant bars (unpaired, two-tailed t test on 2−ΔΔCt values; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). WT, wild type. Sry expression is normally down-regulated as SOX9 levels increase, but it can persist if testis differentiation fails (22, 23). This is consistent with a direct or indirect repressive effect of SOX9 on Sry. At E11.5, Sry mRNA levels were higher than wild-type levels in both Enh13+/− and Enh13−/− XY gonads (168 and 152%, respectively) (Fig. 3B). In contrast, the TES deletion did not significantly alter Sry expression (Fig. 3B), which is expected because Sertoli cell differentiation is proceeding in TES mutants. SF1 is known to interact first with SRY and later with SOX9 to regulate Sox9 expression levels and also many of their downstream target genes (16). We found no significant changes in levels of Sf1 mRNA with any of the enhancer deletions at E11.5 (Fig. 3C). Using Foxl2 as an early marker of granulosa cell differentiation (24), we found that mRNA levels in XX wild-type gonads at E11.5 were 3.6 times as high as those in XY gonads (Fig. 3D). Compared with the latter, Enh13+/− and Enh13−/− XY gonads showed two- and threefold increases, respectively, with the homozygotes having mRNA levels very close to XX control levels. Therefore, Enh13−/− XY gonads reveal an early commitment to the ovarian pathway. There was a 30 to 50% decrease in Sox9 mRNA levels in E11.5 XX Enh13−/− gonads compared to the wild type, as reflected by reduced immunofluorescence for SOX9 protein (fig. S11). These data indicate that Enh13 also plays a role in the very early expression of Sox9 in the XX gonad, consistent both with the small peak seen with ATAC-seq and with occasional reporter activity in the transgenic mouse assays (Fig. 1, B and C, and fig. S7). The sequence of Enh13 is highly conserved among mammals (Fig. 1B) and contains consensus binding sites for transcription factors known to regulate early gonad development and sex determination (fig. S13) (6). Mouse Enh13 contains a single consensus SRY binding site as well as a SOX9 site to which SRY can also bind (Fig. 4A and fig. S13). We performed ChIP-qPCR on E11.5 gonads dissected from Sry-Myc transgenic embryos by using a specific antibody against the MYC tag (22). SRY-MYC–positive gonads had an 11-fold enrichment versus SRY-MYC–negative gonads with primers spanning the SOX9 consensus site and a sixfold enrichment with primers spanning the SRY site, whereas primers against the strongest SRY binding site in TESCO (22) showed fivefold enrichment (Fig. 4, A and B). This reveals the strong binding of SRY to Enh13 at E11.5, with a preference for the SOX9 consensus site, possibly due to the adjacent SF1 binding site. Preferential binding of SRY to Enh13 over TESCO at E11.5 supports the hypothesis that the former is more critical because it initiates up-regulation of Sox9, whereas the latter is secondary. |
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Cruz). (C) β-Gal staining (blue) of E13.5 testes and ovaries from two representative independent stable Enh13 transgenic (Tg) lines. Scale bars, 100 μm.