Initial characterization of CXXC5 as a putative DNA binding protein

Yaşar, Pelin
17β-estradiol (E2), the main circulating estrogen hormone, is involved in the physiological and pathophysiological regulation of various tissue notably mammary tissue functions. E2 is responsible for the cellular proliferation, differentiation and/or death in target tissue. The E2 effect is mediated by the nuclear receptors, estrogen receptor α and β, as ligand-dependent transcription factors. Upon binding of E2, ER is converted to an active form and regulates the expression of target genes primarily through genomic signaling pathways leading to cellular responses. The binding of E2-ER to specific DNA sequences estrogen response elements (EREs) initiates the transcription through the ERE-dependent signaling pathway. The interaction of the E2-ER complex with the other transcription factors that are already bound to their cognate response elements mediates the transcriptional events via the ERE-independent signaling pathway. Previous genome-wide analysis studies of our laboratory suggested that the CXXC5 gene expression is regulated by E2-ERα through the ERE-dependent signaling pathway. CXXC5 is a member of the ZF-CXXC domain protein family that contains a highly conserved CXXC domain and a nuclear localization signal. The ZF-CXXC family proteins, through their CXXC domain, bind to non-methylated CpG dinucleotides in CpG islands of transcriptionally active DNA regions. This binding prevents cytosine methylation and leads to the formation of a nucleation site for the direct or indirect recruitment of histone modifying proteins to DNA for transcription regulation. Although studies on functional features of CXXC5 are scarce, the protein appears to participate as a transcription factor, an epigenetic regulator and/or a co-modulator in the transcriptional regulation of cellular events in response to various signaling pathways. We therefore predict that CXXC5, as a non-methylated CpG dinucleotide binding protein, also plays a fundamental role in E2-mediated cellular events. CXXC5 is located on 5q31.2, oriented on the forward strand and encompasses 35- kb DNA. There are three exons and the parts of the second and third exons generate a 1447 bp long mRNA with 969 bp long open reading frame that encodes a 322 amino-acid long protein with a molecular mass of approximately 33 kDa. To begin examining the structure-function of CXXC5, we initially verified our genome-wide findings that CXXC5 is an estrogen responsive gene using MCF7 cells derived from a breast adenocarcinoma. We found by RT-qPCR that CXXC5 is indeed an E2- and ER-responsive gene. We subsequently cloned the ORF of the CXXC5 transcript into a mammalian expression vector. We examined the synthesis and intracellular location of endogenous and exogenously introduced CXXC5 using western blot, immunocytochemistry and short interfering RNA approaches in cell models. We observed that CXXC5 is synthesized at varying amounts in cell lines of breast carcinomas. We also found that CXXC5 localized in the nucleus and shows a diffuse distribution in interphase appearing to overlap with DNA. However during cell division, CXXC5 displayed a distinct nuclear staining that does not overlap with DNA. This suggests that the intra-nuclear distribution of CXXC5 is cell-cycle dependent. Based on our homology modeling and the presence of CXXC domain we predicted that CXXC5 as other protein members of the ZF-CXXC family is a non-methylated CpG dinucleotide binding protein. To verify this prediction, we analyzed the interactions of CXXC5 and CpG dinucleotide containing DNA fragments by electrophoretic mobility shift assay using whole cell extracts or recombinant protein obtained from a bacterial protein expression system. However, we did not observe any binding of CXXC5 to DNA. These findings suggest either that CXXC5, in contrast to our prediction, is not a DNA binding protein; or that bacterial expression is not an appropriate system for CXXC5 to obtain a functional protein. We are currently addressing these issues using various eukaryotic expression systems. Inclusion of studies on the identification of protein interaction partners of CXXC5 will be an important aspect of future studies aimed at the dissection of structural and functional features of the protein.
Citation Formats
P. Yaşar, “Initial characterization of CXXC5 as a putative DNA binding protein,” M.S. - Master of Science, Middle East Technical University, 2015.