| Description |
ZBTB33 is the first identified zinc finger containing methyl-CpG binding protein (MBP), the cellular function of which is still unclear in both normal and disease state. Gene regulation by ZBTB33 is mediated through recognition of both methylated as well as sequence specific nonmethylated deoxyribonucleic acid (DNA) sites. DNA recognition is followed by recruitment of corepressors resulting in transcription changes. While evidence suggests that transcriptional regulation by ZBTB33 plays a role in cancer, an extensive analysis of the gene targets and signaling pathways regulated by this protein have yet to be investigated. The goal of this research is to begin to determine how ZBTB33 directs disease relevant transcription. To investigate this, a combination of techniques to characterize in cell global gene occupation, DNA methylation status, and transcriptome changes were utilized. Further, the effect of bimodal DNA recognition exhibited by ZBTB33 on transcriptional regulation and biological pathways in cancer were investigated. Powerful techniques have emerged for investigating genome-wide protein-DNA interactions. One such technique incorporates chromatin immunoprecipitation followed by exonuclease digestion and next generation sequencing (ChIP-exo). While this technique maps global gene occupations of transcription factors at high resolution, it also harbors technique specific issues, including the significant generation of artifact peaks, limiting its application. Thus, a method for generating a background control that substantially reduces these artifacts and improves the confidence level in peak identification for ChIP-exo data sets was developed. Application of our improved ChIP-exo method to ZBTB33 revealed additional issues with ChIP-based techniques coupled with next generation sequencing that can affect a number of systems. Thus, efforts to identify and resolve these issues are discussed. Along with ChIP-based methods, whole genome shotgun bisulfite sequencing (WGSBS) and RNA sequencing (RNA-seq) are other powerful methodologies informing on the global DNA methylation status and transcriptome of a cell, respectively. By combining these techniques with ZBTB33 depletion/overexpression studies and phenotypic assays, we found mechanistic details for how ZBTB33 mediates cell-specific cell cycle regulation in a cancerous cell line. Specifically, we identified a mechanism by which ZBTB33 mediates the cyclin D1/cyclin E1/RB1/E2F pathway, controlling passage through the G1-restriction point and accelerating cancer cell proliferation. |
