GATA4 Binding to the Sox9 Enhancer MXYSRa/Enh13 is Critical for Testis Differentiation in Mouse

Overview

Journal Commun Biol

Date 2025 Jan 18

PMID 39827212

Authors

Yuya Ogawa

Iku Tsuchiya

Shogo Yanai

Takashi Baba

Ken-Ichirou Morohashi

Takehiko Sasaki

Junko Sasaki

Miho Terao

Atsumi Tsuji-Hosokawa

Shuji Takada

Affiliations

Soon will be listed here.

Abstract

In mammals, SOX9/Sox9 expression in embryonic gonads is essential for male gonadal sex determination. Multiple enhancers of Sox9 have been identified, of which the mXYSRa/Enh13 enhancer plays a crucial role in mice. SOX9 and SRY binding sites within the enhancer have been identified as functional. Simultaneous deletion of both sites in mice resulted in male-to-female sex reversal. However, the existence of other critical functional sequences remains unclear. This study identified an additional functional sequence by generating mice with partial deletions in mXYSRa/Enh13. Two nucleotide substitutions within the sequence were sufficient for male-to-female sex reversal. In vivo binding assay by CUT&RUN revealed that GATA4 binds to the sequence. In vitro luciferase assay showed that GATA4 promotes the enhancer activity and the substitution of the sequence reduces the effect. Taken together, the functional sequence in mXYSRa/Enh13 is essential for testis differentiation and requires GATA4 binding.

References

Skene P, Henikoff J, Henikoff S . Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc. 2018; 13(5):1006-1019. DOI: 10.1038/nprot.2018.015. View

Munger S, Aylor D, Syed H, Magwene P, Threadgill D, Capel B . Elucidation of the transcription network governing mammalian sex determination by exploiting strain-specific susceptibility to sex reversal. Genes Dev. 2009; 23(21):2521-36. PMC: 2779749. DOI: 10.1101/gad.1835809. View

Ogawa Y, Terao M, Hara S, Tamano M, Okayasu H, Kato T . Mapping of a responsible region for sex reversal upstream of Sox9 by production of mice with serial deletion in a genomic locus. Sci Rep. 2018; 8(1):17514. PMC: 6269501. DOI: 10.1038/s41598-018-35746-0. View

Croft B, Ohnesorg T, Hewitt J, Bowles J, Quinn A, Tan J . Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9. Nat Commun. 2018; 9(1):5319. PMC: 6293998. DOI: 10.1038/s41467-018-07784-9. View

Ogawa Y, Terao M, Tsuji-Hosokawa A, Tsuchiya I, Hasegawa M, Takada S . SOX9 and SRY binding sites on mouse mXYSRa/Enh13 enhancer redundantly regulate Sox9 expression to varying degrees. Hum Mol Genet. 2022; 32(1):55-64. DOI: 10.1093/hmg/ddac184. View

Manuylov N, Fujiwara Y, Adameyko I, Poulat F, Tevosian S . The regulation of Sox9 gene expression by the GATA4/FOG2 transcriptional complex in dominant XX sex reversal mouse models. Dev Biol. 2007; 307(2):356-67. PMC: 2020840. DOI: 10.1016/j.ydbio.2007.04.040. View

Langmead B, Salzberg S . Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4):357-9. PMC: 3322381. DOI: 10.1038/nmeth.1923. View

Bouma G, Washburn L, Albrecht K, Eicher E . Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice. Proc Natl Acad Sci U S A. 2007; 104(38):14994-9. PMC: 1986601. DOI: 10.1073/pnas.0701677104. View

Narimatsu K, Iida A, Kobayashi T . Palatoplasty for the Patient With Campomelic Dysplasia-Report of a Case and Review of the Literature. Cleft Palate Craniofac J. 2021; 59(1):132-136. DOI: 10.1177/1055665621992654. View

10.

Calvache C, Vasquez E, Romero V, Hosomichi K, Pozo J . Novel SRY-box transcription factor 9 variant in campomelic dysplasia and the location of missense and nonsense variants along the protein domains: A case report. Front Pediatr. 2022; 10:975947. PMC: 9716274. DOI: 10.3389/fped.2022.975947. View

11.

Li S, Hannenhalli S, Ovcharenko I . De novo human brain enhancers created by single-nucleotide mutations. Sci Adv. 2023; 9(7):eadd2911. PMC: 9931207. DOI: 10.1126/sciadv.add2911. View

12.

Bouma G, Albrecht K, Washburn L, Recknagel A, Churchill G, Eicher E . Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells. Development. 2005; 132(13):3045-54. DOI: 10.1242/dev.01890. View

13.

Gonen N, Futtner C, Wood S, Garcia-Moreno S, Salamone I, Samson S . Sex reversal following deletion of a single distal enhancer of . Science. 2018; 360(6396):1469-1473. PMC: 6034650. DOI: 10.1126/science.aas9408. View

14.

Kent J, Wheatley S, Andrews J, Sinclair A, Koopman P . A male-specific role for SOX9 in vertebrate sex determination. Development. 1996; 122(9):2813-22. DOI: 10.1242/dev.122.9.2813. View

15.

Kuroki S, Matoba S, Akiyoshi M, Matsumura Y, Miyachi H, Mise N . Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a. Science. 2013; 341(6150):1106-9. DOI: 10.1126/science.1239864. View

16.

Zhu Q, Liu N, Orkin S, Yuan G . CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis. Genome Biol. 2019; 20(1):192. PMC: 6734249. DOI: 10.1186/s13059-019-1802-4. View

17.

Skene P, Henikoff S . An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife. 2017; 6. PMC: 5310842. DOI: 10.7554/eLife.21856. View

18.

Mansour S, Hall C, Pembrey M, Young I . A clinical and genetic study of campomelic dysplasia. J Med Genet. 1995; 32(6):415-20. PMC: 1050480. DOI: 10.1136/jmg.32.6.415. View

19.

Ramirez F, Ryan D, Gruning B, Bhardwaj V, Kilpert F, Richter A . deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016; 44(W1):W160-5. PMC: 4987876. DOI: 10.1093/nar/gkw257. View

20.

Kim G, Sock E, Buchberger A, Just W, Denzer F, Hoepffner W . Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development. J Med Genet. 2015; 52(4):240-7. DOI: 10.1136/jmedgenet-2014-102864. View