טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentWijeweera Andrea
SubjectGnRH Regulates Gonadotropin Subunit Gene Transcription
Through its Effects on Chromatin
DepartmentDepartment of Biology
Supervisor Professor Philippa Melamed
Full Thesis textFull thesis text - English Version


Abstract

The synthesis of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) are regulated by the gonadotropin releasing hormone (GnRH). GnRH was shown to regulate transcription of these genes through inducing histone deacetylase displacement and histone acetyltransferases recruitment, suggesting that GnRH may regulate transcription through altering the chromatin structure. We hypothesized that transcriptional activation of the subunit genes by GnRH involves the induction of specific histone modifications previously found implicated in yeast and mammalian transcriptional regulation.


We demonstrate that the chromatin structure is involved in regulating basal activity of all three gonadotropin subunit genes. The αGSU promoter adopts an open conformation due to low nucleosome occupancy, histone hyperacetylation and high levels of H3K4me3. The increased accessibility facilitates binding of RNAPII and gene-specific transcription factors, resulting in high basal gene expression. While both β-subunit genes adopt a closed conformation, these genes still contain histone modifications typically associated with active genes, albeit at significantly lower levels. The LHβ promoter also contains slightly higher levels of these modifications and exhibits increased promoter accessibility to transcription factors and RNAPII as compared to the FSHβ promoter, in accordance with its higher basal activity, indicating that the chromatin likely plays a role in fine tuning basal gene expression. 


Upon GnRH treatment, there is up-regulation of αGSU expression and induction of both β-subunit genes. ChIP studies demonstrate that GnRH induces and/or enriches the level of H3K4me3 on all promoters, with the induction of H3K4me3 occurring due to the increased recruitment of the menin-MLL complex. Targeting of this complex likely occurs through its interaction with ERα and/or SF-1. We show a novel role for this modification in regulating gonadotropin subunit gene transcription, as inhibition of H3K4me3 resulted in significant decreases in gonadotropin subunit gene mRNA levels. Our findings also suggest that the H3K4me3 catalyzed by the menin-MLL1/2 complex is likely independent of prior H2BK120ub. On the αGSU promoter, GnRH induces hyperacetylation of H3 at K9, K18 and K27 which may be dependent on prior H3S10p, a modification catalyzed by MSK1, whose activation is GnRH-induced. H3S10p is essential for GnRH-induced upregulation of αGSU gene expression.


These results demonstrate that basal and GnRH induced gonadotropin subunit gene expression is regulated by H3K4me3. GnRH-induced upregulation of the αGSU also requires H3S10p, which may crosstalk with H3 acetylation. Collectively, we demonstrate the novel role of chromatin in mediating basal and GnRH-induced transcriptional activation of these genes through inducing various histone modifications.