The phenotype of absence: investigating the role of SRY and USP9Y in DNA damage response pathways

The Y chromosome is the smallest chromosome of the human male genome and with the fewest number of protein-coding genes. It is known for its role in male sex determination and regulation of autosomal gene expression. Alterations of the Y chromosome are linked to infertility, various malignancies and pathologies. In particular, loss of the Y chromosome (LOY), a frequent event during aging, is associated with chromosomal instability, increased cancer predisposition with poor clinical outcomes and altered responses to anticancer treatments. Previous studies in our laboratory demonstrated that LOY in non transformed cells causes defects in DNA damage response (DDR) pathways activation. In this project, I investigated which are the Y-linked genes involved in the establishment of DDR defects associated with LOY phenomenon in both cancer and non-transformed cell lines. To identify possible candidates, we initially exploited RNA-sequencing analyses performed in parental and Y-negative non transformed ARPE-19 cell line before and after etoposide treatment. These studies revealed that in parental ARPE-19 cells only nine Y-linked genes are expressed and that three of them, USP9Y, DDX3Y and EIF1AY, are upregulated in response to genotoxic stress. In addition, after etoposide treatment, also SRY gene bocomes expressed. Among the upregulated Y encoded genes, we decided to focus on SRY (sex determining region on the Y) and USP9Y (ubiquitin specific peptidase 9 Y-linked). The SRY protein initiates male sex determination. In addition, its involvement in DDR, male tumours, and potential oncogenic effects have been reported. USP9Y, a deubiquitinating enzyme (DUB) that removes ubiquitin from target substrates, is clinically relevant for spermatogenesis and male infertility. It also functions as a tumour suppressor, involved in cell proliferation and apoptosis. Its homologue, USP9X, is also implicated in regulating proliferation, apoptosis, and drug resistance. Therefore we silenced SRY and USP9Y in ARPE-19 cells and assessed their involvment in DDR defects associated with LOY phenomenon. We found that SRY depletion did not fully recapitulate the DDR defects observed after Y chromosome loss, indicating that additional Y-linked genes might contribute to the LOY phenotype. We therefore silenced USP9Y and demonstrated that also its depletion partially mimicked LOY effects. Of note, both Y-negative and USP9Y silenced ARPE-19 cells showed reduced etoposide-induced PARP1 cleavage, suggesting lower apoptosis and increased treatment resistance. Accordingly, colony forming assays confirmed improved etoposide survival for both Y-depleted and USP9Y silenced cells, while SRY loss didn’t significantly impact cellular survival upon this treatment. Similar experiments were then performed in the lung cancer cell line A549, to determine whether these genes have the same effects on non-transformed and cancer cell lines. In these cells, both SRY and USP9Y depletion caused DDR defects, some of which were consistent with those observed in ARPE-19 whereas others appeared to be specific for this cell line. As in ARPE-19, USP9Y loss reduced PARP1 cleavage and promoted cell survival after etoposide treatment also in A549, while SRY depletion didn’t demonstrate any effect in this context. Importantly, during these studies we observed that USP9Y depletion reduces the proliferation of both unstressed ARPE-19 and A549 cells. Since in both these cell lines the absence of USP9Y promoted the accumulation of p53 and p21waf1 that might induce cell cycle arrest, we silenced USP9Y and p53 in different combinations and we checked cell growth before and after etoposode treatment. We found that loss of p53 partally rescues the growth defect induced by USP9Y absence. These findings therefore indicate that both SRY and USP9Y participate to the DDR although their loss does not fully replicate the DDR defects seen with Y-depleted cells.