Establishment of cell lines with inducible expression OF shRNA for an estrogen responsive gene

Karakaya, Burcu
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 CXXC5 as a member of zinc-finger CXXC domain protein family, binds to non-methylated CpG dinucleotides. Methylation of CpG islands found in enhancer and promoter regions is thought to silence gene transcriptions. Binding of this family proteins to non-methylated CpGs is suggested to have a role in transcription by preventing DNA methylation. Although studies on CXXC5 are limited, CXXC5 appears to participate in cellular processes as a transcription factor, co-regulator and/or epigenetic factor. To address the role of CXXC5 in E2-mediated cellular events, we aimed 1) to identify protein partners and 2) to define cellular function of CXXC5. Since, proteins perform their functions within the context of interacting proteins in a spacio-temporal manner, we initially decided to use a yeast-two-hybrid (Y2H) service for the identification of putative CXXC5 protein partners followed by a mammalian-two-hybrid system (M2H) for verification. However, of the reported interacting partners identified with Y2H, we were not able to verify any protein as the CXXC5 interacting partner with M2H. Based on these results, we decided to use the BioID approach that allows the identification of interacting proteins through the biotin tagging of interactors. Our ongoing studies suggest that BioID can indeed be used for the identification of CXXC5 interactors. In studies we performed in parallel to Y2H to address the role of CXXC5 in E2-mediated cellular events, we wanted to generate cell lines in which CXXC5 was stably overexpressed or silenced. However, due to cellular death; we failed to generate stable cell lines. Our failure necessitated the use of an inducible expression system to generate cell lines that allow us to conduct functional studies on CXXC5. The system was expected to provide us maintainable cell lines containing on/off switch for CXXC5 expression. For this, we used pINDUCER system, which provides a tetracycline (Tet) inducible expression of target gene. For this purpose, we initially used an shRNA approach to specifically down-regulate CXXC5 synthesis. Although we have succeeded the generation of monoclones, we could not decrease CXXC5 synthesis. Studies for obtaining overexpressed or silenced CXXC5 has been continuing with different cell lines and expression systems.


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 ...
Assessment of 17beta-estradiol-estrogen receptor alpha complex-mediated changes in genome-wide methylation and gene expression profiles
User, Sırma Damla; Muyan, Mesut; Department of Biology (2016)
17β-estradiol (E2), the most potent estrogen hormone, induces cellular responses primarily through Estrogen Receptor-alpha (ERα), which is a transcription factor. Interfering E2 signaling indicates that E2 is mitogenic for cells, exemplified by MCF7 cells derived from breast adenocarcinoma, synthesizing ERα endogenously. Studies used exogenous expression of ERα in ERα-negative cell lines to examine structural/functional properties of the receptor. What was unexpected from these studies is the observation th...
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 ...
The regulation of the CXXC5 gene expression
Yaşar, Pelin; Muyan, Mesut; Department of Molecular Biology and Genetics (2021-1-19)
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...
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 ...
Citation Formats
B. Karakaya, “Establishment of cell lines with inducible expression OF shRNA for an estrogen responsive gene,” M.S. - Master of Science, Middle East Technical University, 2018.