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.