Indiana University-Purdue University Indianapolis (IUPUI) / LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.

Indiana University-Purdue University Indianapolis (IUPUI) / Several common diseases such as atherosclerosis, post-angioplasty restenosis, and graft vasculopathies, are associated with the changes in the structure and function of smooth muscle cells. During the pathogenesis of these diseases, smooth muscle cells have a marked alteration in the expression of many smooth muscle-specific genes and smooth muscle cells undergo a phenotypic switch from the contractile/differentiated status to the proliferative/dedifferentiated one. Serum response factor (SRF) is the major transcription factor that plays an essential role in coordinating a variety of transcriptional events during this phenotypic change. The first goal of my thesis studies is to determine how SRF regulates the expression of smooth muscle myosin light chain kinase (smMLCK) to mediate changes in contractility. Using a combination of transgenic reporter mouse and knockout mouse models I demonstrated that a CArG element in intron 15 of the mylk1 gene is necessary for maximal transcription of smMLCK. SRF binding to this CArG element modulates the expression of smMLCK to control smooth muscle contractility. A second goal of my thesis work is to determine how SRF coordinates the activity of chromatin remodeling enzymes to control expression of microRNAs that regulate the phenotypes of smooth muscle cells. Using both mouse knockout models and in vitro studies in cultured smooth muscle cells I showed how SRF acts together with Brg1-containing chromatin remodeling complexes to regulate expression of microRNAs-143, 145, 133a and 133b. Moreover, I found that SRF transcription cofactor myocardin acts together with SRF to regulate expression of microRNAs-143 and 145 but not microRNAs-133a and 133b. SRF can, thus, further modulate gene expression through post-transcriptional mechanisms via changes in microRNA levels. Overall my research demonstrates that through direct interaction with a CArG box in the mylk1 gene, SRF is important for regulating expression of smMLCK to control smooth muscle contractility. Additionally, SRF is able to harness epigenetic mechanisms to modulate expression of smooth muscle contractile protein genes directly and indirectly via changes in microRNA expression. Together these mechanisms permit SRF to coordinate the complex phenotypic changes that occur in smooth muscle cells.