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Link to original content: http://pubmed.ncbi.nlm.nih.gov/38555298/
A conserved NR5A1-responsive enhancer regulates SRY in testis-determination - PubMed Skip to main page content
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. 2024 Mar 30;15(1):2796.
doi: 10.1038/s41467-024-47162-2.

A conserved NR5A1-responsive enhancer regulates SRY in testis-determination

Denis Houzelstein  1   2 , Caroline Eozenou  3   4   5 , Carlos F Lagos  6   7 , Maëva Elzaiat  3   4 , Joelle Bignon-Topalovic  3   4 , Inma Gonzalez  4   8 , Vincent Laville  4   9   10 , Laurène Schlick  3   4 , Somboon Wankanit  3   4   11 , Prochi Madon  12 , Jyotsna Kirtane  13 , Arundhati Athalye  12 , Federica Buonocore  14 , Stéphanie Bigou  15 , Gerard S Conway  16 , Delphine Bohl  15   17 , John C Achermann  14 , Anu Bashamboo  3   4 , Ken McElreavey  18   19
Affiliations

A conserved NR5A1-responsive enhancer regulates SRY in testis-determination

Denis Houzelstein et al. Nat Commun. .

Abstract

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Organization of the SRY locus in human.
a A sequence fragment spanning 80 kilobases (GRC38h38.p13-chrY: 2786855-2867268) from SRY to RPSY4. The intergenic region is predominantly formed of highly repetitive elements. b Zoomed-in view of a subregion from a covering the sequence from SRY to the beginning of the repetitive elements (GRC38h38.p13-chrY:2,786,855- 2,794,240). E800 corresponds to the region sequenced in 46,XY individuals with unexplained gonadal dysgenesis described in Fig. 3. E250 corresponds to the 250 bp fragment used for the luciferase assay described in Fig. 4E. The light blue, dark blue, and red triangles represent consensus binding sites for GATA4, WT1, and NR5A1, respectively. Turquoise indicates repetitive sequences. Open arrows represent SRY and RNASEH2CP1, while black boxes represent the RPS4Y1 exons. The transcription Start Site (TSS) of SRY is indicated by an arrow. c Consensus binding sites for GATA4, WT1, and NR5A1 predicted by Matinspector (from the Genomatix suite).
Fig. 2
Fig. 2. Sequence conservation at the SRY locus.
a The profile of accessible chromatin, determined by DNase-seq, was downloaded from the Encode project (https://www.encodeproject.org/, experiment ENCSR729DRB, embryonic human testis). It reveals two distinct regions of accessible chromatin, one encompassing the SRY gene and the other corresponding to the E250 region, centered on the predicted NR5A1 binding site (NR5A1_bs). The graphical representation of the human SRY locus is shown below, in phase with the DNase-seq profile, and the corresponding number of base pairs is indicated on the right. GATA4 (light blue), WT1 (dark blue), and NR5A1 (red) Matinspector predicted binding sites are indicated as in Fig. 1b. E800 corresponds to the region sequenced in 46,XY individuals with unexplained gonadal dysgenesis. E250 corresponds to the 250 bp fragment used in the luciferase assay (Fig. 4e). Repetitive sequences are shown in turquoise, the SRY gene, and RNASEH2CP1 pseudogene Open Reading Frame as open arrows. b DNA fragments from eutherian representative species, aligned to the 7046 bp human sequence shown in a. The sequences homologous to E800 are shown in black. The conservation of these sequences across a diverse range of species, including sloth, human, springhare, pig, bat, and tapir, indicates that this region was already present in their last common ancestor. Similarly, the presence of an NR5A1 predicted binding site in a homologous position shown by a red line in these species suggests a role for this NR5A1 binding site early in eutherian radiation. The total number of bases aligned to the human sequence for each species is indicated on the right. c The species presented in Fig. 2b were selected to represent diversity in the eutherian radiation. An estimate of sequence conservation is given. Where sequences could be aligned, a 100% conservation means that a nucleotide is conserved in all the 18 sequences. A conservation of 50% would mean that for a given aligned nucleotide, it is conserved in only half of the species. The SRY gene and E800 sequences both show the highest percentage of sequence conservation in the region.
Fig. 3
Fig. 3. Pedigrees of a familial case of 46,XY disorder of sex development (DSD).
a, b Two pedigrees illustrating the two branches of a family from the same community, presenting with 46,XY gonadal dysgenesis. Arrows indicate the three individuals for whom the whole genome sequence was obtained. c Distribution of phenotypes, incidence of gonadal tumors, and assigned sex of individuals with the SRY variant.
Fig. 4
Fig. 4. In silico and in vitro analysis of E250 function.
a Alignment of the sequence flanking the NR5A1-binding site (GRCh38-chrY: 2,792,790-2,792,804), in the reference genome (Reference), mutated in the familial case (Variant-1), in the sporadic case (Variant-2), and in a negative control with four substitutions disrupting the core sequence (∆NR5A1). The NR5A1 consensus binding site is shown above for comparison, featuring the core element from +1 to +6 and the flanking sequence from −1 to −3. b–d Schematic representation of NR5A1 DNA-binding domain (DBD) interactions with E250 variants based on the crystal structure of NR5A1 bound to the inhibin-A promoter. Protein residues of NR5A1 are depicted in green, while the DNA backbone is shown in orange. Hydrogen bond contacts are represented as red dashed lines. b Structural model of wild-type NR5A1 bound to DNA. The two paired binding site residues of interest are shown, based on the sequences in panel A (A-1/T- 1 and G + 3/C + 3). NR5A1 binds to DNA primarily as a monomer. The protein P-box region (codons 31 to 35) interacts directly with the core binding motif (+1 to +6), whereas the A-box protein region (codons 89 to 92) interacts with the flanking DNA sequence (−1 to −3). c, d Structural models of wild-type NR5A1 bound to variant response elements associated with testicular dysgenesis. In Variant-1 (panel c), the A > G purine to purine change at nucleotide −1 in the flanking sequence (shown in cyan, with corresponding reverse strand T > C change) affects interactions between the DNA and A-box residues changing the H-bond pattern between DNA and basic residues R87, R89, R92, R94, and Y99 of NR5A1. In contrast, with Variant-2 (panel d), the G > C purine to pyrimidine change at nucleotide +3 (shown in magenta, with corresponding reverse strand C > G change) affects interactions between the DNA and P-box. There is disruption of critical H-bond interactions that would typically occur between NR5A1 codon K38 and the DNA amine group of G9, and between codon E31 with the amine group of C22; furthermore, the amine group of codon K34 is neutralized by H24 and E31 in NR5A1. These changes induce a displacement of both “A-box” and “P-box” helices that may influence binding affinity or kinetics. e Transient gene transfection assay showing activation of a luciferase reporter construct containing the wild-type or variant E250 response element co-transfected with or without NR5A1 in HEK293T cells (12 technical replicates, outliers identified by the interquartile Range (IQR) method and then removed, with comparison performed using the Wilcoxon rank-sum exact test (p value = 1.379e-08****) in R.
Fig. 5
Fig. 5. Sertoli-like cell differentiation from hiPSCs.
a Differentiation kinetics of human hiPSCs derived from a healthy 46,XY male, starting with hiPSC, and sequential alteration of the culture medium (M1, M2, and M3) to induce differentiation into Sertoli-like cells. For greater clarity, the x-axis starts after 30 h in M1. The labels M1-36h, M2-06h, M2-12h, M2-24h, M2-48h, and M3-48h indicate the time cells spent in specific media, while the labels 36, 42, 48, 60, 72, 84, 108, and 132 h indicate time elapsed since the initiation of differentiation, triggered by the replacement of mTeSR with M1 medium. The expression levels of key genes, SRY (blue), SOX9 (red), and WNT4 (green) were quantified using qRT-PCR with the ∆∆CT method. Normalization was performed using the 18S rRNA RPL19 gene, and the end of M1 served as the calibration point. b Comparison of SRY, SOX9, and WNT4 expression between wild-type and mutant E250-∆33 hiPSC cell lines. The expression levels were quantified using qRT-PCR with the ∆∆CT method using the wild-type condition as the calibrator for the visual representation. Gene expression is visually represented with wild-type clones shown in dark and mutant clones light blue for SRY, in red and light brown for SOX9, and in dark and light green for WNT4. The data were pooled with two to five biological replicates performed, with each experiment having three to six technical replicates. The statistical analysis was performed using a linear mixed model. At the initial timepoints (M1-36h, M2-06h), data were shown within a box, to highlight a 40 to 70% reduction in relative expression of SRY in mutant cells compared to wild-type control cells (detailed in the Supplementary Information file).
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