Methylation-Sensitive Regulation of TMS1 Expression andChromatin Structure by the Ets Factor GABP Público

Lucas, Mary Ekasha (2009)

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Epigenetic silencing involving the aberrant DNA methylation of promoter-associated CpG islands is one mechanism leading to the inactivation of tumor suppressor genes in human cancers. However, the molecular mechanisms underlying this event remain poorly understood. TMS1/ASC is a novel proapoptotic signaling factor that is subject to epigenetic silencing in human breast and other cancers. The TMS1 promoter is embedded within a CpG island that is unmethylated in normal cells and is spanned by three DNaseI hypersensitive sites (HS). Silencing of TMS1 in cancer cells is accompanied by local alterations in histone modification, remodeling of the HS and hypermethylation of DNA.

In this study, we identified two roles for the GA-Binding Protein complex (GABP) in the regulation of transcription from the TMS1 locus. Initially, GABP was identified as a methylation-sensitive complex that bound a 55bp intronic element within HS2 in vitro and in vivo and functioned as an activator of transcription. In reporter assays the HS2 element conferred a 3-fold enhancement in TMS1 promoter activity, which was dependent on both intact GABPα binding sites and the presence of GABP proteins (GABPα and GABPβ1) in trans. Accordingly, downregulation of GABPα led to a concomitant decrease in TMS1 expression.

In addition, we identified a role for GABP in the maintenance of transcriptional competency at the TMS1 locus through the regulation of histone acetylation. Inhibition of GABP binding at the TMS1 locus through deletion of GABP binding sites correlated with a decrease in histone H3 acetylation at lysine 9 and 14. These data indicate that the intronic HS2 element acts in cis to maintain transcriptional competency and hyperacetylated histones at the TMS1 locus and that this activity is mediated by the ets transcription factor, GABP.

In conclusion, this project demonstrates GABP is necessary for the activation of transcription from the TMS1 locus and the maintenance of a hyperacetylated chromatin state. Therefore, the loss of GABP binding at the TMS1 locus could be one mechanism leading to the loss of acetylated histones, the propagation of heterochromatic histone modifications and silencing of TMS1 expression in cancer.

Table of Contents

I. List of Figures

II. Chapters

Chapter 1: Introduction

Chapter 2: GABPα Binds the TMS1 Locus at HS2 in vivo in a Methylation Sensitive Manner and acts a Positive Regulator

Chapter 3: Impact of GABP Chromatin Structure at the TMS1 Locus

Chapter 4: Discussion

III. Literature Cited

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