Martin J Walsh, PhD

• 2010
  Senior Scholar Award in Aging
  

The Modulation of Chromatin Structure and Function in Transcription

The capacity to reprogram gene expression programs determine the fate of cells to self renew, differentiate or terminate. The transcription of genetic information from DNA is the fundamental process that regulates gene expression and requires elaborate and complex signals necessary to overcome the normally repressive state of chromatin. Our laboratory has focused on understanding the mechanisms that regulate gene expression through processes that recognize and establish epigenetic character in chromatin necessary to facilitate or repress gene transcription. Much of our recent work has been in close collaboration with Ming-Ming Zhou to adopt innovative approaches that will understand the molecular, structural and biochemical basis for epigenetic control of gene expression. Recently, our studies solved the molecular structure and the biochemical function of the SWIRM domain, a phylogenetically conserved structure common in many chromatin-associated proteins. Much of our ongoing effort has been to combine structure -guided analysis that will utilize chemistry-based experimental designs to determine the molecular and cellular function of chromatin proteins in vivo in a more effective manner. Future studies plan to exploit the physical structure of proteins that interact with DNA, RNA and modified histones and to use chemical probes to assess their biochemical and cellular function in a native cellular environment. Ongoing studies also exploit the use of chromatin immunoprecipitation sequencing (ChIP-Seq) and high -density sequencing maps to determine the global impact on cellular chromatin. Below is a brief summary of ongoing projects in the laboratory.

Regulation of chromatin structure by human CUTL1 transcription factor-
Our focus has been directed on two fundamental transcription factors that play key roles in both oncogenic transformation and during development called the CCAAT displacement protein/cut homologue (CUTL1) and zinc finger protein 217 (ZNF217/zfp217). CUTL1 in man and cux in mouse are essential for development and self -renewal in various tissue compartments in metazoan vertebrates. CUTL1 is also a key determinant in promoting tumor cell migration and metastasis. We have previously shown that CUTL1 can mediate the acetylation and methylation of nucleosomal histones through the differential recruitment and of histone acetyltransferase (HAT), histone deacetylase (HDAC), and histone lysine metyltransferase (HMT) activities. We have also demonstrated that CUTL1 is a substrate of many of these enzymes that determine the function of CUTL1. Although, transcription factors have the ability to bind DNA they typically lack the capacity to navigate chromatin structure necessary to access cognate DNA sequences. Many transcriptional co-regulators provide the function to recognize and bind post-translational -modified nucleosomal histone. When tethered to co-regulators, transcription factors attain the ability to associate with highly ordered chromatin structure and impose their regulatory function. Our ongoing studies are investigating the role novel nuclear co-regulators and their conserved protein domains that can "read" the histone code by binding modified histone residues that mark functional domains within chromatin. Identification and analysis by ChIP-Seq of transcriptional co-regulators for CUTL1 and the histone marks they impose will help us determine the "visual scope" and native context for CUTL1 occupation within the human genome.

Perpetuation of cellular self -renewal by the ZNF217 oncogene-
ZNF21/zfp217 is an important oncogene in many cancer types. It impacts cell physiology markedly by shifting the apoptotic threshold of cancer cells causing resistance to the chemotherapeutical agent doxorubicin and contributing to telomere stability and immortalization under certain experimental conditions. While recent observations are clearly relevant to the understanding of ZNF217's role(s) in cancer, they represent an indirect effect through ZNF217's aggregate activity on the large number of genes that it targets. Little is known about how ZNF217 operates at the level of individual genes, i.e., about its principal mode(s) of molecular action as transcription factor. We have now demonstrated that ZNF217 forms a nuclear complex that can modify histones. Specifically, we documented H3K4me3 demethylation; H3K9 methylation; and H3K27 methylation. We identified five nuclear proteins contained in the ZNF217 complex, namely Jarid1b/Plu-1, a histone H3 lysine 4 (H3K4) tri-methyl demethylase; G9a, a principal euchromatic H3K9 methylase EZH2, a H3K27 methylase associated with the Polycomb Repressive Complex 2 (PRC2); LSD1, a H3K4 demethylase; CtBP1 and CoREST, which are both transcriptional co-repressors. Our studies illuminate the view that ZNF217 adopts a dynamic configuration of chromatin modifying enzymes to adapt to the localized chromatin environment. In collaboration with Dr. Gail Mandel's laboratory (Vollum Institute), we have now directed the ablation of the ZNF217 orthologue zfp217 in mouse embryonic stem cells

Epigenetic programming of Polycomb through long non -coding RNAs-
More recent studies investigate the regulation of the Polycomb group (PcG) and Trithorax (Trx) proteins in exerting gene control through the coordination of binding between long non-coding RNAs, histone lysine methylation and ubiquitination during early embryonic development and during oncogenesis. We have recently directed our effort to understand the role of long non-coding RNAs (ncRNAs) to mediate the function of Polycomb Repressive Complexes  (PRCs) 1 and 2. Transcription is pervasive through out the mammalian genome, however, most transcripts are non-coding in the genome but have been thought to impose an architectural function in chromatin Our recent studies have shown that ncRNAs are an instructional component of chromatin that mediates the function of PRC1 and PRC2 to further impose histone modifications for epigenetic gene silencing. Current studies by RNA ChIP-Seq are evaluating the role of ncRNAs that mask the human genome to coordinate PRC function during development and in human disease.