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Human Sex Reversal Tied to Enhancer Mutations Near SOX9 Gene

NEW YORK (GenomeWeb) – Researchers in Australia have uncovered three enhancers that regulate a key sex determination gene in humans.

While X and Y chromosomal makeup broadly determines sex at conception, other factors like the Y-linked gene SRY also influence sex determination. SRY upregulates the gene that encodes the SOX9 transcription factor, high levels of which are needed for testis development. Copy number alterations affecting these regions may also influence whether someone develops as male or female, despite their chromosomal makeup, but the genetic basis of many disorders of sex development remains unknown.

Researchers from the Murdoch Children's Research Institute in Melbourne further examined the genomic regions surrounding SOX9 for clues of factors that influence sex determination among humans. As they report in Nature Communications this week, they identified three SOX9 enhancers that, when deleted or duplicated, led to sex reversal.

"This study is significant because in the past, researchers have only looked at genes to diagnose these patients, but we have shown you need to look outside the genes to the enhancers," first author Brittany Croft said in a statement.

She and her colleagues first analyzed stretches of DNA in the regulatory region upstream of SOX9 that had previously been associated with two disorders of sex development, 46,XY and 46,XX.

With data from a cohort of 44 patients with disorders of sex development, the researchers zeroed in on a 5.2-kilobase region that was duplicated in two 46,XX patients. They subcloned this region into five overlapping fragments and used a luciferase reporter assay to find that four of the five fragments exhibited enhancer activity when co-transfected with SOX9 and SF1, which acts with SRY to activate an SOX9 enhancer. In particular, a 1,524-base pair fragment, which the researchers dubbed Sex Reversal Enhancer-A (eSR-A), exhibited the strongest response. This fragment, they noted, contained one SRY/SOX9 and two SF1 consensus-binding motifs.

They likewise investigated another upstream regulatory region of SOX9, known as RevSex, to narrow a region thought to contain an enhancer signal to 24 kilobases by analyzing how a duplication in a patient with 46,XX ovotesticular disorder of sex development overlapped with previously amassed data. This 416-base pair region, which they called Sex Reversal Enhancer-B (eSR-B), includes an SOX9 binding motif, a p300 binding site, and a DNase I hypersensitive site.

The researchers noted that as eSR-A and eSR-B enhancers are duplicated in patients with SRY-negative 46,XX disorders of sex development, they could be activated in the absence of SRY. They conducted a bioinformatics screen to search for SRY-responsive conserved enhancer marks upstream of SOX9 to find a highly conserved 1,259-base pair fragment harboring a DNase I hypersensitive site and a putative SRY binding site.

This fragment, they reported, was significantly activated by co-transfection with SRY and SF1 or SOX9 and SF1, but did not respond to SOX9 alone. They dubbed this enhancer Alternation Long-Distance Initiator (eALDI) of SOX9.

"We discovered three enhancers that together ensure the SOX9 gene is turned on to a high level in an XY embryo, leading to normal testis and male development," senior author Andrew Sinclair from MCRI said in a statement.

Through a series of luciferase activity assays, the researchers developed a model of how SOX9 is activated. According to that, SRY and SF1 are expressed early in the human fetal gonad in XY individuals. With the eALDI enhancer, they then initiate SOX9 expression. The expression of the SOX9 gene is upregulated and maintained not only by the SOX9 protein itself and SF1, but also by the enhancers eSR-A, eSR-B, and eALDI.

"These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD," the researchers wrote.

Their discovery could also help to improve the diagnosis of disorders of sex development. Only about 40 percent of patients receive a genetic diagnosis at the moment, and "this study shows that these newly defined regions should be included in any diagnostic assays for unexplained cases of DSD," they wrote.