טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYosefzon Yahav
SubjectThe Regulation of TET Proteins and Their Role in Controlling
Expression of the LH Beta Gene
DepartmentDepartment of Biology
Supervisor Professor Philippa Melamed
Full Thesis textFull thesis text - English Version


Abstract

The pituitary gland’s gonadotrope cells synthesize the gonadotropins, FSH and LH. These hormones play fundamental roles in gonad development and function. The genes encoding them are quiescent soon after birth and their expression is re-initiated at puberty following GnRH stimulation. Therefore changes at the chromatin level are required in order to overcome their repressed state. Repressed chromatin is characterized by methylcytosine at CpG dinucleotides, which could be converted by the Tet enzymes to hydroxymethylcytosine, an active feature of transcription. In this work we aimed to explore possible roles of DNA modifications in mediating activation of these genes. For this purpose we used an immature gonadotrope cell line, which does not express the gonadotropin genes and thereby represents their repressed state, and a mature gonadotrope cell line in which the LH and FSH are expressed. We found that the LHβ promoter has a CpG island (CpGI) whose DNA methylation status is related to its levels of expression. We also found that both Tet1 and Tet2 are expressed in gonadotopes and are involved in regulating LHβ expression. Tet2 binds the LHβ CpGI and apparently activates transcription, while Tet1 appears to suppress expression of this gene. We found that Tet1 expression is downregulated in the mature gonadotropes, and also following GnRH treatment. We found that Tet1 has a CpGI 22kbp upstream of its TSS which is methylated in mature gonadotropes, in accordance with its reduced expression, but not in immature gonadotropes in which it is readily expressed. Moreover, its unmethylated state is mediated by Tet2 which binds this CpGI. The distal location of the Tet1 CpGI indicates that this region might function as an enhancer. Indeed, we found that it is enriched with the enhancer marker H3K4me1, and also that it is transcribed and the ncRNA levels correlate with those of the Tet1 transcript. Therefore it is possible that Tet1 expression is regulated through the production of this ncRNA, whose expression is affected by methylation of the upstream CpGI, or vice versa. Taken together, our results suggest that LHβ expression is determined by DNA modifications at its own promoter and also at the Tet1 distal regulatory element, and that these modifications have a pivotal role in regulating expression of both genes. These findings further our understanding of the how the chromatin landscape plays a role in determining the differential regulation of gene transcription, and specifically the role of the Tet enzymes.