Abstract A47: Investigating Zbtb protein recognition of epigenetically modified DNA

Abstract

Abstract Methylation of CpG dinucleotides is essential for genomic stability, control of gene expression and the regulation of chromatin structure in normal eukaryotic cells. Aberrant alterations in these genomic methylation patterns have been linked with many diseases, including cancer. This correlation has prompted the need to better understand the regulatory mechanisms of DNA methylation in gene transcription. The Zbtb family, consisting of Zbtb33, Zbtb4 and Zbtb38, is a set of methyl-CpG binding proteins (MBPs), which recognize both methylated as well as sequence-specific non-methylated DNA sites through conserved Cys2His2 zinc fingers. DNA recognition subsequently recruits chromatin remodeling machinery to the target site resulting in chromatin compaction and gene silencing. While sufficient evidence suggests that the Zbtb proteins play a role in cancer, an extensive analysis of the gene targets and signaling pathways regulated by each protein has yet to be investigated. Here we utilize an interdisciplinary approach combining cellular biology, molecular biology and structural biology to determine the epigenetic targets regulated by the Zbtb MBPs and to identify the protein region(s) necessary for high-affinity DNA recognition. Utilizing western blot analysis, we have determined that the three Zbtb proteins exhibit differential endogenous expression levels in a variety of cancer cell lines. Further, ChIP qRT-PCR analysis demonstrated that the occupancy of Zbtb33 at various methylated and sequence-specific genes was highly variable between the different cancer cell lines. Combined, these results suggest that the Zbtb proteins likely mediate differential signaling pathways in different cancers, possibly by targeting specific regulatory gene elements. Mapping of the global gene occupation for Zbtb33 in various cancer cell lines is in progress. Additionally, we have designed multiple protein constructs around the conserved Cys2His2 zinc finger DNA binding region as well as additional zinc fingers located in the C-terminal regions of Zbtb4 and Zbtb38, as the functions of the latter have not yet been identified. The constructs have been analyzed by solution NMR and EMSA to determine whether they are amenable for further structural analysis and to characterize their DNA binding interactions. This multidisciplinary approach provides the means for advancing our understanding of the molecular basis by which these proteins interpret and translate epigenetic signals in the cancerous state. Citation Format: Tommy W. Terooatea, Maya J. Pandya, Alan C. Chugg, Bethany A. Buck-Koehntop. Investigating Zbtb protein recognition of epigenetically modified DNA. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A47.