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Learn More. Cell fate decisions require appropriate regulation of key genes. Sox9a 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 Mb gene desert upstream of Sox9.

Our data is consistent with the time-sensitive activity of SRY and indicates a strict order of enhancer usage. Enh13 is conserved and embedded within a The regulation of genes with important roles in embryonic development can be complex, involving multiple, often redundant enhancers, repressors, and insulators 12.

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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 12.

The developing gon comprise an interesting system in which to explore questions of gene regulation during development 3.

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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 gon 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 rather than granulosa cells typical of ovaries 7.

Sox9the 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 their maintenance 4 — 6. Both gain and loss of function studies in mouse and human demonstrate that Sox9 plays a key role in testis determination 8 — The only enhancer known to be relevant for expression in Sertoli cells was TES, a 3.

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We therefore used several unbiased approaches to systematically screen for additional gonad enhancers upstream of mouse Sox9. We first made use of DNaseI-seq data obtained with E From 33 putative enhancers we chose only those positive at both stages for in vivo validation by transgenic assays 14 enhancers, Fig.

S1, S2 and methods. Most putative enhancers discovered by DNaseI-seq were evident in the E Chromatin immunoprecipitation-seq ChIP-seq was also performed for H3K27ac, a histone modification that marks active enhancers fig. A A schematic representation of the gene desert upstream of the mSox9 gene and the location of the putative enhancers identified by ATAC-seq and DNaseI-seq that were screened in vivo using transgenic reporter mice.

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Enhancers that did not drive gonad-expression of LacZ are shown in grey. Enhancers that drove testis-specific and ovary-specific LacZ expression are shown in blue and pink, respectively. DNaseI-seq black on E Peaks correspond to nucleosome depleted regions, and are marked by black box if they are ificantly enriched compared to flanking regions as determined by MACS, and present in at least two biological replicates.

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The grey box overlaying each peak indicates the cloned fragment. Green lines represent sequence conservation between mouse, human, rhesus, cow and chicken sequence conservation tracks obtained from UCSC. All 16 putative enhancers were cloned upstream of an hsp68 minimal promoter and the reporter gene LacZ, and used to generate transgenic mice 219 Table 1.

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For initial screens we performed transient analyses at E The remaining four showed gonad expression and these constructs were re-injected to generate stable lines in order to better study their activity in both males and females during development. This may be due to Enh8 being taken out of its original genomic context; notably ATAC-seq reveals a much stronger peak in granulosa cells compared to Sertoli cells fig. S1 and S5A. To test this candidate, we used genome editing to delete Enh However, Enh14 deletion did not alter expression of Sox9, its target gene Amhor Foxl2a marker of granulosa cells, in E S5Dindicating that Enh14 has a redundant role, at least in the embryo.

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S1 and S6A. S1, S7A. ATAC-seq data from E S1suggesting that it opens co-incident 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 Enh Irrespective of whether this was carried out on a TES mutant background or by itself it always led to XY female development Fig. Similar analysis of XX Enhdeleted gon did not show any obvious phenotype fig.

Turquoise and purple arrows represent the external and internal sgRNAs used to delete Enh13, respectively. Black arrows represent the PCR primers used to genotype Enh13 deleted embryos and mice.

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C Immunostaining of E Sex reversed gon are indistinguishable from WT XX gon, while the heterozygous deletion does not appear to alter testis morphogenesis. As before, XY heterozygotes presented as normal fertile males whereas homozygotes showed full male-to-female sex reversal fig. S8, S 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.

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Therefore, E Deleting one or two copies of TES had relatively little effect at E This again supports the conclusion that Enh13 plays a more ificant role than TES during early gonadal development. Sample size represents the of individuals and is indicated below each genotype. Dark grey bars: XY; light grey bars: XX. Sry expression is normally downregulated as SOX9 levels increase, but it can persist if testis differentiation fails 22 This is consistent with a direct or indirect repressive effect of SOX9 on Sry.

At E Using Foxl2 as an early marker of granulosa cell differentiation 24mRNA levels were 3. These data indicate that Enh13 also plays a role in the very early expression of Sox9 in the XX gonad, consistent with both the small peak seen with ATAC-seq and with occasional reporter activity in the transgenic mouse assays Fig. The sequence of Enh13 is highly conserved among mammals Fig. S13 6.

The bovine Enh13 is indicated by the grey box at Cow Bostau8 chr 60,, Y Axis s represent counts. The input tracks represent sequencing re of chromatin input. The bSox9 gene is indicated by the arrow and is kb downstream to the left of Enh Unlike several other gonadal enhancers, Enh13 appears well conserved, has a clear role in mice to initiate upregulation of Sox9 expression, in response to SRY activity, and it may contribute to its maintenance.

This makes it very likely to play a similar role in humans given its location within the XYSR region 20 It is clear from our data and that of others that the upstream regulatory region of Sox9 is very complex. Our screens using gonadal cells revealed 33 potential enhancers distributed over 1.

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Transgenic assays used to test the most promising 16, revealed four that gave expression in the gon, whereas the majority did not. In addition, several putative enhancers, notably Enh4, 5, 8, and 9, appear open in both granulosa and Sertoli cells, with Enh8 even more so in the former. These could contribute to the low level of Sox9 seen in supporting cell precursors, but they may also represent sequences required to repress Sox9which might not be detected by transgenic reporter assays.

It is therefore remarkable to see that deleting Enh13 alone phenocopies the loss of Sox9 itself within the supporting cell lineage 9 Substantial evidence points to the time-dependent action of SRY on Sox9.

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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, begin to act in a more redundant fashion, although it is conceivable that each 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.

We thank members of our labs for advice, support, and helpful comments. M was funded by the Northwestern University School of Medicine. Author contribution: N. G and R. B deed the study. S and D. S helped with analysing the reporter mice.

W performed cytoplasmic and pronuclear zygote injections. G performed the rest of the experiments. B analysed and interpreted the and wrote the manuscript. All authors reviewed and added input to the manuscript.