Mediation of gene expression via histone-DNA interactions: nucleosome positioning on the PHO5 gene of Saccharomyces cerevisiae

Abstract

The vast majority of genetic information in eukaryotic cells must be packaged in the highly repressive environment of chromatin to fit within the confines of the cell nucleus. To fully understand how genes are expressed the chromatin environment in which all transcription processes occur should not be overlooked. This study will focus on developing an understanding of how chromatin structure at the level of the nucleosome must become ordered to result in coordinated gene expression. The PHO5 gene of the yeast Saccharomyces cerevisiae will be utilized for this research. The PHO5 gene encodes a secreted acid phosphatase that is activated in conditions of low phosphate. In the repressed secreted acid phosphatase that is activated in conditions of low phosphate. In the repressed state, four nucleosomes are positioned along the promoter region causing repression by blocking access of the basal transcription machinery to the minimal promoter element, while a critical nucleosome-free region is maintained to allow binding of the transcriptional activator Pho4p to the promoter region. Exactly how this nucleosome array is formed is not known. This document contains the development of an in vitro chromatin reconstitution system used to study the mechanism of gene activation and repression in the context of chromatin. This system, created entirely from the yeast Saccharomyces cerevisiae, was used to recreate the repressed state of the PH05 gene as demonstrated by nucleosome positioning and repression of transcription in vitro. The presence of proper nucleosome positioning in an in vitro setting show that intrinsic DNA properties are sufficient to properly position nucleosomes on the PHO5 promoter. Moreover, it was demonstrated that variability in general isotropic DNA bending could largely account for nucleosome positioning on the PH 05 promoter. This work reveals how histone-DNA contacts create an initial layer of gene regulation that we are now only beginning to understand.