Identification and regulation of p53 target genes in primary human epidermal keratinocytes

Abstract

BIOCHEMISTRY

IDENTIFICATION AND REGULATION OF P53 TARGET GENES IN PRIMARY HUMAN EPIDERMAL KERATINOCYTES

KRISTY L. SCHAVOLT

Dissertation under the direction of Professor Jennifer A. Pietenpol The p53 tumor suppressor gene is mutated in half of all human cancers. p53 mediates the cellular response to a variety of cell stress signals through transcriptional regulation of target genes that play roles in cell cycle arrest, DNA repair, and apoptosis. Over one hundred target genes are directly regulated by p53, though mechanisms regarding p53 regulation of gene transcription are not fully understood. To gain further insight to the processes of p53 target gene regulation, the dissertation research described herein resulted in the identification of novel targets of p53, as well as analysis of factors that dictate the ability of p53 to selectively regulate target genes in response to genotoxic stress. The role of p53 family members p63 and p73 in p53-mediated signaling pathways was investigated as well. A primary human keratinocyte model system was utilized to avoid genetic and epigenetic alterations found in established cell lines. To identify novel target genes, fragments of p53-bound DNA isolated by chromatin immunoprecipitation (ChIP) were functionally tested for p53 regulation using a yeast screen. To complement the ChIP/yeast screen, microarray analysis was performed to identify changes in gene expression upon ectopic expression of p53 and deltaNp63alpha. In these studies, Ras-related associated with diabetes (RRAD), modulator of apoptosis 1 (MOAP-1), and zinc finger protein 90 (ZFP90) were identified as potential p53 transcriptional targets. Finally, timing of transcription factor binding to target gene regulatory regions was examined using ChIP. At target gene consensus binding sites, p53 occupancy increased and concomitantly, deltaNp63alpha occupancy decreased. Correspondingly, expression of a panel of known p53 target genes was increased by p53 and decreased by deltaNp63alpha in microarray studies. Additionally, neither the presence of constitutively bound p53 nor deltaNp63alpha dictates the constitutive binding of RNA polymerase II (pol II) to target gene proximal promoters. Rather, the location of the consensus binding site (promoter or intron) dictates the constitutive binding of pol II. This research allows for a more complete understanding of p53 transcriptional regulation of target genes at the levels of promoter occupancy and gene expression, as well as a potential role of deltaNp63alpha in p53-regulated signaling. Understanding mechanisms of p53 target gene regulation is essential for comprehending how alterations in p53 signaling result in increased tumorigenesis.

Approved___Jennifer Pietenpol________ Date_____11.30.06______