|M.Sc Student||Rabinovich-Tsukerman Anna|
|Subject||Regulation of Tet1 and Tet2 Expression in the Gonadotrope|
|Department||Department of Biology||Supervisor||Professor Philippa Melamed|
|Full Thesis text|
DNA methylation is an epigenetic mark that plays a crucial role in gene regulation during development. Mechanisms of DNA demethylation are not fully understood, however the Tet deoxygenases that catalyze oxidation of 5mC to 5-hydroxymethylcytosine are likely involved. We found that Tet enzymes are expressed in gonadotropes at different levels depending on the degree of cell differentiation. We also found that the number of the gonadotrope cells in immature mice was higher than in mature mice, and that Tet1 was expressed at a higher level in these cells, but it was downregulated following their final differentiation. We discovered that the Tet1 isoform expressed in these gonadotropes lacks the CXXC zinc finger domain at the N terminus due to utilization of a novel downstream transcription start site. Additionally we have shown that both GnRH and estradiol, which comprise physiological regulators of the reproductive axis, regulate Tet expression. In αT3-1 gonadotrope-precursor cells and in primary gonadotrope cells, the expression of Tet1 and Tet2 is altered by GnRH, leading to a decrease in Tet1 and an increase in Tet2 mRNA levels. To clarify the pathways involved, we treated αT3-1 and/or LβT2 mature gonadotrope cells with PMA to activate the protein kinase C (PKC) pathway, the calcium ionophore ionomycin, or forskolin to activate the cAMP/protein kinase A (PKA) pathway. PMA had no effect on Tet1 mRNA levels, but did increase those of Tet2, while ionomycin decreased levels of both transcripts, with a stronger effect on Tet1. In contrast, forskolin repressed Tet1 and activated Tet2 expression. Confirmation of the role of the cAMP/PKA pathway in mediating the distinct GnRH effects on each of the Tet genes, was seen by pre-incubation of cells with the PKA inhibitor, H89 which abolished both the GnRH inhibitory effect on Tet1 and its stimulatory effect on Tet2. Our results suggest that the divergent effects of GnRH on expression of the Tet enzymes are mainly through the cAMP/PKA pathway, although PKC activation may also increase Tet2 transcription. We have thus shown a regulatory role for GnRH on expression of these enzymes which likely helps shape gonadotrope function, and initiated clarification of the intracellular mechanisms involved. We also found that estradiol represses both Tet enzymes in poorly differentiated gonadotrope cells, but only Tet1 in more differentiated cells. Moreover we show that the ESR1 receptor binds upstream of the Tet1 gene to elicit this effect directly. Accordingly, after ovariectomy, removal of this negative feedback led to an increase in Tet1 levels, while expression of Luteinizing Hormone β subunit (LHβ), which is repressed by Tet1, decreased. This negative correlation between Tet1 and LHβ mRNA levels was seen also after castration, although at the time point tested, Tet1 levels were reduced and LHβ increased. This is likely due to the removal of the negative feedback on GnRH which represses Tet1 and also stimulates LHβ. Our findings provide better understanding of the regulation of Tet enzymes which play a critical role in the regulation of gonadotrope differentiation and expression of the LHβ gene.