Date

2021-1-19

Author

Yaşar, Pelin

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17β-estradiol (E2) is the main circulating estrogen hormone in the body and is involved in the physiological and pathophysiological regulation of various tissue notably mammary tissue functions. E2 is responsible for cellular proliferation, differentiation, and/or death in target tissues. Our previous microarray studies suggested that expression of CXXC5 is regulated by E2-ERα through ERE-dependent signaling pathway and I verified that the CXXC5 transcript levels are augmented in response to E2. As a member of the ZF-CXXC domain protein family, CXXC5 harbors a highly conserved CXXC domain and nuclear localization signal. It is known that functionally characterized ZF-CXXC domain protein family members bind to preferentially non-methylated CpG dinucleotides in CpG islands of transcriptionally active DNA regions via their CXXC domains, and we showed that CXXC5 also binds to non-methylated CpG dinucleotides. Cytosine methylation is prevented due to this binding, and a nucleation site formation is induced for the direct/indirect recruitment of transcription co-regulators, histone-modifying proteins, which leads to the regulation of transcription. According to the limited studies on CXXC5, it appears to be that CXXC5 participates in the cellular events as an epigenetic regulator and/or co-modulator in response to various signaling pathways. However, the mechanisms of how the CXXC5 gene expression is mediated remain unknown. In my doctoral studies, I showed the expression and the synthesis of CXXC5 is E2- and ERα-dependent. I found that an intronic ERE sequence in the CXXC5 locus, and I showed that this de novo ERE is bound by ERα in vitro and in cellula. In addition, I showed that the binding of the E2-ERα complex is functional and resulted in the transcriptional activation using reporter enzyme assays. To understand how this distally located ERE participates in the regulation of CXXC5 expression regulation; first I wanted to identify the CXXC5 promoter. Since there are 14 annotated transcript variants for CXXC5, I foresaw that the identification of the main CXXC5 variant(s) expressed at the highest amount in MCF7 cells as our cell model is necessary for the promoter analyses studies. Therefore, after the identification of the primary CXXC5 transcript (CXXC5-TV2), I conducted 5’Rapid Amplification of cDNA Ends (5’RACE) studies to uncover the transcription start site(s) (TSSs) of the CXXC5-TV2 to characterize the core promoter elements which are generally found in proximity to the TSSs. Next, I performed luciferase reporter assays to locate the core promoter elements of the CXXC5. I showed that the CXXC5 promoter region resides within the first exon of the CXXC5-TV2, and the expression of CXXC5 is driven by a CGI promoter (CpG Island Promoter). After identifying the promoter region, I continued with promoter pull-down studies to characterize the putative CXXC5 promoter interactor proteins. Of the identified proteins by Liquid chromatography-tandem mass spectrometry (LC-MS/MS), I validated the binding of the ELF1 (E74 Like ETS Transcription Factor 1), MAZ (Myc-associated zinc finger protein), and RB1 (Retinoblastoma-associated protein) to the CXXC5 promoter. I also found the sequence motifs for DNA binding of ELF1 and MAZ in the CXXC5 promoter. I verified that ELF1 and MAZ contribute to the regulation of CXXC5 expression using endogenous and heterologous gene expression approaches. In summary, I found that the expression, and consequently, the synthesis of CXXC5 are regulated by E2-ERα signaling through an intronic ERE. I located the CXXC5 promoter region and identified the several transcription factors engaged with the promoter, and verify that these transcription factors are involved in the gene expression regulation of CXXC5. The findings presented in this dissertation could provide a basis for understanding the regulation of an E2-responsive gene CXXC5, therefore, could provide a better understanding of the E2-ER signaling actions in physiological and/or pathophysiological conditions.

Subject Keywords

Estrogen Signaling, CXXC5, Promoter, CpG island, Expression Regulation

URI

https://hdl.handle.net/11511/89649

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Graduate School of Natural and Applied Sciences, Thesis