The goal of this project is to define genetic/epigenetic mechanisms that regulate the fine balance between tissue homeostasis and cancer. Neural stem cells (NSCs) differentiate into multiple lineages of the brain, and perturbations in this process can lead to glioma, a brain cancer that is notoriously difficult to treat. A frequent genetic lesion in glioma is deletion of 1p36. CHD5 was discovered as a tumor suppressor encoded within this chromosomal region, and high CHD5 expression predicts better patient survival following glioma treatment. This proposal seeks to build upon these initial discoveries by determining the mechanism whereby CHD5 regulates chromatin to affect global gene expression cascades that control NSC differentiation/fate and to define how perturbation of these CHD5-mediated pathways affects glioma development. The goal of Aim 1 is to determine the mechanism whereby Chd5 regulates chromatin marks and gene expression cascades in NSCs. This will be accomplished by: A) identifying Chd5-interacting proteins using immunoprecipitation and mass spectrometry, B) defining global gene expression and covalent histone modification patterns by performing RNA-sequencing and ChIP-sequencing in NSCs from Chd5-compromised mice, and C) identifying functional motifs of Chd5 required for its interactions with other proteins and for evoking covalent histone modifications critical for transcriptional regulation of its target genes, and by assessing how tumor-derived CHD5 mutations obstruct these capabilities. The goal of Aim 2 is to define the role of Chd5 in regulating NSC differentiation/fate. This will be accomplished by: A) determining how Chd5 deficiency affects stem cell populations, cell cycle dynamics, and differentiation using flow cytometry, B) defining the temporal pattern of expression and subcellular localization of Chd5-interacting proteins and target genes using quantitative PCR, western blotting, and immunofluorescence in NSCs from Chd5-lacZ reporter mice during the differentiation process, and C) assessing how motif-specific and tumor-derived mutations affect Chd5's ability to regulate NSC differentiation/fate. The goal of Aim 3 is to assess the consequence of perturbing Chd5 and the pathways it regulates on glioma formation in vivo. This will be accomplished by: A) determining how Chd5 deficiency and lesions in Chd5-modulated pathways affect gliomagenesis using orthotopic transplantation, B) assessing how closely Chd5-mediated alterations in the mouse parallel human glioma by comparing global patterns of histone marks and gene expression of Chd5-compromised NSCs to those of patient glioma samples, and C) defining the effect that motif-specific and tumor-derived CHD5 mutations have on gliomagenesis. This project will use unique models for Chd5 to further define the biological role of Chd5- mediated chromatin dynamics in neurogenesis and in glioma development. In addition, this work will elucidate genetic/epigenetic processes that impact glioma, thereby revealing cancer-specific vulnerabilities that may offer pharmacological interventions for treating this currently incurable malignancy.
This project will determine how the machinery that packages our DNA controls the behavior of cells of the brain, and will elucidate how defects in this process lead to brain cancer. We discovered that CHD5-a protein that packages DNA-plays an important role in normal brain cells that when absent, leads to glioma. Here we propose to advance these findings by determining how CHD5 normally prevents glioma, as this work has the potential to reveal new ways of treating this tumor that given the prevalence of CHD5 mutations in a variety of other human cancers, may lead to breakthroughs for treating other malignancies as well.
