Integrated Chromatin Analyses Offer Insights Into Trans-factor Function In Cancer Cell Lines

Abstract

<p>Understanding the mechanisms whereby the sequence of the human genome is interpreted into diverse cellular phenotypes is a critical endeavor in modern biology. A major determinant of cellular phenotype is the spatial and temporal pattern gene expression, which is regulated in part by epigenomic properties such as histone post-translational modifications, DNA methylation, chromatin accessibility and the 3-dimensional architecture of the genome within the nucleus. These properties regulate the dynamic assembly of transcription factors and their co-regulatory proteins upon chromatin. To properly understand the interplay between the epigenomic framework of a cell and transcription factors, integrated analysis of transcription factor-DNA binding, chromatin status, and transcription is required. This work integrates information about chromatin accessibility, as measured by DNaseI hypersensitivity, transcription factor binding, as measured by chromatin immunoprecipitation, and transcription, as measured by microarray or transcriptome sequencing, to further understand the functional role of two important transcription factors, the androgen receptor (AR) and CTCF, in cancer cell line models. Data gathered from a prostate cancer cell line model demonstrate that the AR does not exclusively bind accessible chromatin upon ligand-activation, and induces significant changes in chromatin accessibility upon binding. Regions of quantitative change in chromatin accessibility contain motifs corresponding to potential collaborators for AR function, and are also significantly associated with AR-regulated transcriptional changes. Furthermore, base pair resolution of the DNaseI cleavage profile revealed three distinct patterns of AR-DNA interaction, suggesting multiple modes of AR interacting with the genome. A novel role for the nuclear receptor REV-ERB&#945; in AR-mediated transcription was explored within the same model system. Though preliminary, results thus far indicate that REV-ERB&#945; is required for AR-induced increases in target gene transcription in a manner that is likely dependent on HDAC3. Genetic knockdown of REV-ERB&#945; resulted in notable changes in chromatin accessibility around AR-target genes both before and after AR activation. The function of CTCF was interrogated using stable knockdown in a breast cancer cell line model. CTCF knockdown led to widespread changes in chromatin accessibility that were dependent on DNA sequence. Further analysis suggested that AP-1 and FOXA1 are involved in CTCF function. Together, the work presented in this dissertation offers novel insight into the behavior of two critical transcription factors in cancer cell lines, and describe a framework of analysis that can be extended and applied to any transcription factor within any desired cellular context.</p>