The regulation of the CXXC5 gene expression

Yaşar, Pelin
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.


Functional importance of CXXC5 in E2-driven cellular proliferation
Razizadeh, Negin; Muyan, Mesut; Department of Biology (2019)
17β-estradiol (E2) as the main circulating estrogen hormone has an important role in the regulation of various tissues including mammary tissue. E2 effects target tissue functions by binding to the nuclear receptors, ERα and β. ERs regulate the expression of target genes. Previous studies conducted in our laboratory indicate that one of these estrogen responsive genes is CXXC5 which is regulated by ERα. CXXC5 has a highly conserved zinc-finger CXXC domain, which makes it a member of zinc-finger CXXC domain ...
Molecular mechanism of estrogen-estrogen receptor signaling.
Yaşar, P; Ayaz, G; User, Sd; Güpür, G; Muyan, Mesut (2016-12-05)
17 beta-Estradiol (E2), as the main circulating estrogen hormone, regulates many tissue and organ functions in physiology. The effects of E2 on cells are mediated by the transcription factors and estrogen receptor (ER)alpha and ER beta that are encoded by distinct genes. Localized at the pen-membrane, mitochondria, and the nucleus of cells that are dependent on estrogen target tissues, the ERs share similar, as well as distinct, regulatory potentials. Different intracellular localizations of the ERs result ...
Demiralay, Öykü Deniz; Muyan, Mesut; Department of Biology (2022-8-22)
17β-estradiol (E2) is the main estrogen in circulation and has many physiological effects on various tissues, including the mammary tissue. CXXC5 is an estrogen-responsive gene product that binds to nonmethylated CpG dinucleotides on DNA. CXXC5 synthesis shows fluctuation in the cell cycle. This led to our prediction that the level of CXXC5 synthesis is regulated through the cell cycle. To test this prediction, I investigated the synthesis of CXXC5 in cell cycle-synchronized cells for every 6h up to 36h. I ...
Cloning and initial protein characterization of an estrogen responsive gene: YPEL2
Güpür, Gizem; Muyan, Mesut; Department of Biology (2014)
17β-estradiol (E2), the main circulating estrogen in the body, is involved in physiological regulation of many tissue and organ functions, including mammary tissue. E2 is also involved in target tissue malignancies. E2 regulates cellular proliferation, differentiation and death in target tissues. The lasting effects of E2 on cells are mediated by estrogen receptor and β that are the products of distinct genes and act as transcription factors. Upon binding to E2, the activated ER regulates the expression of ...
Establishment of cell lines with inducible expression OF shRNA for an estrogen responsive gene
Karakaya, Burcu; Beklioğlu, Meryem; Department of Biology (2018)
Estrogen hormones, primarily 17β-estradiol (E2) as the primary circulating estrogen, are involved in the homeodynamic regulation of various tissues/organs including mammary gland within which estrogen receptor α (ERα) conveys E2 signaling. The binding of E2 to ERα activates the receptor to regulate estrogen responsive gene expressions. Previous microarray and gene expression studies of our laboratory indicate that CXXC5 is an estrogen responsive gene regulated by ERα. Our ongoing studies also indicate that ...
Citation Formats
P. Yaşar, “The regulation of the CXXC5 gene expression,” Ph.D. - Doctoral Program, Middle East Technical University, 2021.