VALIDATING DIFFERENTIALLY EXPRESSED GENES IN MET-2 DELETION MUTANTS AND IDENTIFICATION OF GENES RESPONSIBLE FOR SPERMATOGENIC DEFECTS REGULATED BY H3K9 METHYLATION

Abstract

One important mechanism of genetic regulation is through post-translational histone modifications. A histone methyltransferase known as MET-2 is an enzyme responsible for such modifications. Specifically, MET-2 is responsible for dimethylation of lysine 9 of histone H3 (H3K9me2), thus promoting heterochromatin structure and resulting in repression of gene expression (Bessler et al., 2010). Consequently, the absence of H3K9me2 leads to the overexpression of various genes, threatening germline integrity, which is vital for proper embryonic development (Delaney et al., 2019). The function of H3K9me2 has been previously studied in oogenesis. However, these germline effects may also impact spermatogenesis in C. elegans. This is evidenced by data indicating met-2 deletion males have significantly reduced fertility. The goal of this research is to explore if misregulation of specific genes is responsible for the spermatogenic defects seen in met-2 mutants. RNAseq was performed on control and met-2 male germ lines and data was collected on differentially expressed genes. Based on previous characterization for functions in meiotic progression or fertilization I am investigating eight of the met-2 differentially expressed genes that were found to be upregulated. My first project goal was to validate upregulation of transcripts in met-2 males compared to control by performing qPCR to analyze mRNA levels of the genes of interest. Analysis of qPCR results did not verify differential expression of the genes of interest in male enriched samples of met-2 versus control worms. The second project goal was to explore in more depth the function of one gene of interest, rmd-1, and how misregulation of gene expression contributes to the spermatogenic defects seen in met-2 mutants. The gene rmd-1 is involved in spindle organization and microtubule attachment to the kinetochore. Preliminary data of RNAi knockdown of rmd-1 shows reduced percent embryonic viability, meanwhile having no effect on brood size. Although both gametogenesis and fertilization occur in rmd-1 knockdown animals, these results indicate that proper expression of rmd-1 may be necessary for the production of gametes of high quality. I have also begun examining spermatogenesis-specific effects of rmd-1 RNAi knockdown via male Embryonic Viability Assays and DAPI staining of the dissected male gonads. Thus far, rmd-1 RNAi knockdown males may have an extended transition zone. More replicates of male EVAs, as well as additional staining and imaging of the gonads in the future will help further elucidate details of the possible role of rmd-1 in spermatogenic defects regulated by H3K9 methylation.