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Peter N. Lewis |
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In eukaryotes replication and transcription occur in spite of the highly compact (1000 times more than protein free DNA) structure of eukaryotic chromosomes. While most of chromosomal DNA is packaged into nucleosomes, it is clear that transcriptionally active genes exist in a less structured condition than transcriptionally inert genes. The molecular mechanism for the formation and maintenance of this "active" state, which is likely a primary determinant of selective gene expression and control, is still uncertain and is the principal focus of our studies. Transcriptionally active chromatin is characterized by core histone hyperacetylation, a modest depletion in the linker histones responsible for the formation of higher order structures, the presence of histone acetyltransferases and structurally altered nucleosomes. The projects described below have as their ultimate goal the elucidation of the molecular mechanisms underlying the in vivo modulation of chromatin structure with particular reference to selective gene expression.
Chromatin Assembly and Remodelling How chromatin is assembled in vivo is poorly understood. Extracts from Drosophila, Xenopus and yeast have an assembly activity which requires ATP and can assemble core histones onto DNA with physiological spacing. These extracts have several components. Two are histone chaperones called the nucleosome assembly protein (NAP1) and Anti-silencing function (ASF1). These proteins together with histones and DNA produce uniformly spaced nucleosomes which are closely spaced along the chromatin fibre. NAP1 forms complexes with the histone octamer through the amino terminal tails of the core histones. Even in chromatin there is a detectable association with the tails and thus NAP1 and ASF1 may act to destabilize the nucleosome and facilitate sliding along the DNA as evinced by a demonstrated remodelling activity. We are interested in the role that these proteins play in chromatin assembly and are exploring the possibility that they modulate the access of other molecules such as histone modifying enzymes to the histone N termini in the nucleus.
Histone DeacetylasesCertain highly evolutionarily conserved lysine residues in the core histones are subject to reversible acetylation catalyzed by the action of histone acetyltransferases and deacetylases. Some of these enzymes are well known transcription factors such as GCN5 and RPD3 which in the yeast Saccharomyces cerevisiae ultimately regulate aspects of cellular metabolism. Histone hyperacetylation is associated with transcriptional activity while histone hypoacetylation often but not always correlates with transcriptional quiescence. We are investigating structure-function relationships in the histone deacetylase family. We hope to understand the basis of their sequence specificity and in vivo regulation. Studies are ongoing to determine the three dimensional structure of one member, HOS3, which has the unusual property , for a deacetylase, of having catalytic activity without the need for other protein cofactors. |
Selected Publications |