OR19-01 Srd5a1 Undergoes Distinct Changes in Methylation and Is Differentially Expressed During Pre-Pubertal Development in the Ovary and Hypothalamus

Abstract

Abstract Disclosure: B. Bar-Sadeh: None. P. Melamed: None. The steroidogenic enzyme, 5α-reductase-1, is responsible for conversion of testosterone to dihydrotestosterone, and production of other 5α-reduced steroids and their derivatives, including in the brain. We reported previously that Srd5a1 levels are reduced in mice hypothalamic preoptic area (POA) and ovaries following early-life immune stress, which appeared to underlie delayed puberty onset and faster depletion of the ovarian reserve. This drop in ovarian Srd5a1 expression was concomitant with hypermethylation at two CpGs in its first intron, and also at the orthologous site in buccal DNA of women who experienced childhood immune challenge1. We thus hypothesized that this region of Srd5a1 acts as a transcriptional enhancer and is regulated by DNA methylation. Here we examined Srd5a1 expression and methylation at these CpGs across normal mouse development and in cultured cells. Ovarian Srd5a1 increased 8-fold and CpG methylation decreased by as much as 75% between PND10-30. During this time estradiol (E2) levels rise, and E2 exposure increased Srd5a1 mRNA levels in primary ovarian cells and KK-1 cell line. Chromatin immunoprecipitation (ChIP) confirmed binding of estrogen receptor to the locus of the CpGs, and identified an adjacent region strongly enriched with H3K4me1, characteristic of transcriptional enhancers. In the POA, in sharp contrast, Srd5a1 mRNA levels decreased by 70% between PND7-10 and then stayed quite constant, with little correlation to CpG methylation levels. The neonatal drop in POA Srd5a1 coincides with an increase in glucocorticoid (GC) levels, and we had seen lower Srd5a1 expression in mice following early-life stress, we thus treated GT1-7 hypothalamic neuronal cells with dexamethasone. This treatment reduced Srd5a1 expression, and ChIP confirmed that the GC receptor binds the enhancer locus. Srd5a1 expression was 12-fold higher in the ovarian KK1 cells compared to the neuronal GT1-7 cells, in accordance with much higher methylation levels in the latter. We thus used these cells to investigate the role of this CpG methylation, using gRNAs targeting dCas9-TET1 for demethylation in GT1-7 cells, or dCas9-DNMT3 for inducing methylation in KK1 cells. In GT1-7 cells, methylation at the first CpG dropped >50%, though was unaltered at the second CpG, and no effect was seen on Srd5a1 expression or its response to E2. In contrast, methylation in KK1 cells increased at the second CpG only, by more than 2.5 fold, and this abolished the response to E2. These distinct changes in Srd5a1 expression during development, together with our findings in cultured cells suggest a cell-specific GC-activated repression of Srd5a1 in the POA, and its E2-activated stimulation in the ovaries regulated by methylation at one of these CpGs. Reference: 1. Bar-Sadeh, B., Amichai, O.E., Pnueli, L., Begum, K., Leeman, G., Emes, R.D., Stöger, R., Bentley, G.R., and Melamed, P. (2022). BMC Biol. 20, 11. Presentation Date: Saturday, June 17, 2023