Abstract
Sex development, a complex and indispensable process in all vertebrates, has still not been completely elucidated, although new genes involved in sex development are constantly being discovered and characterized. Chromobox Homolog 2 (CBX2) is one of these new additions and has been identified through a 46,XY girl with double heterozygous variants on CBX2.1, causing Differences of Sex Development (DSD). The mutated CBX2.1 failed to adequately regulate downstream targets important for sex development in humans, specifically steroidogenic factor 1 (NR5A1/SF1). To better place CBX2.1 in the human sex developmental cascade, we performed siRNA and CBX2.1 overexpression experiments and created a complete CRISPR/Cas9-CBX2 knockout in Sertoli-like cells. Furthermore, we deployed Next Generation Sequencing techniques, RNA-Sequencing and DamID-Sequencing, to identify new potential CBX2.1 downstream genes. The combination of these two next generation techniques enabled us to identify genes that are both bound and regulated by CBX2.1. This allowed us not only to expand our current knowledge about the influence of CBX2.1 in human sex development, but also to advance our insight in the mechanisms governing one of the most important decisions during embryonal development, the commitment to either female or male gonads.
Highlights
Sex development, a complex and indispensable process in all vertebrates, has still not been completely elucidated, new genes involved in sex development are constantly being discovered and characterized
The Next Generation Sequencing (NGS) approach returned 2176 significantly regulated genes under Chromobox Homolog 2 (CBX2).1 knockdown (Fold Change (FC) > 1, False Discovery Rate (FDR) < 0.05) and 146 significantly regulated genes under CBX2.1 overexpression, 54 of which show a significant change in expression under both treatments
CBX2.1 dependent genes show enrichment for other developmental process like kidney development, CNS development, spleen development and bone development. This is in accord with what has previously been shown in mice with M33 (CBX2 homologue) ablation, which showed skeletal malformations and defects in the splenic and adrenal development, human mutants do not seem to recapitulate these defects[3]
Summary
A complex and indispensable process in all vertebrates, has still not been completely elucidated, new genes involved in sex development are constantly being discovered and characterized. The combination of these two generation techniques enabled us to identify genes that are both bound and regulated by CBX2.1 This allowed us to expand our current knowledge about the influence of CBX2.1 in human sex development, and to advance our insight in the mechanisms governing one of the most important decisions during embryonal development, the commitment to either female or male gonads. Transactivation experiments revealed that the variant CBX2.1 does not adequately stimulate the expression of the target gene NR5A1 ( known as SF1), which is essential for human sex development[5] This placed CBX2.1 upstream of SRY in the human sex development cascade, which is in accordance with the mouse experiments and the known expression window of CBX2 in the early male gonad (week 7 of gestation), prior to testis determination[6]. Transactivation studies showed that the variant CBX2.2 failed to regulate the expression of several genes including EMX2, an important factor for bipotential gonadal development
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