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Chromatin Mediated Transcriptional Repression

All cells of an organism contain the same genetic information (DNA), but each cell expresses only a fraction of the genes they contain. There is increasing evidence that the transcription of DNA into RNA is often controlled by the way the DNA is packaged into chromatin.

In Heterochromatin, DNA is tightly packaged by proteins, and most genes cannot normally be expressed. In Euchromatin, the DNA is less tightly packaged and its packaging state can be modified by proteins. They either loosen it to allow, or tighten it to prevent, expression of a gene. The HP1 Protein is known to be associated with heterochromatin. We have been studying the structure and interactions of HP1 to understand better its role in chromatin packaging and gene silencing. We have shown that HP1 contains two structural domains, an N-terminal chromatin modifier (chromo) domain and a C-terminal shadow chromo domain. These domains are found in a large number of other proteins, many probably having similar roles.

Structural comparisons of the chromo domain from HP1, whose structure we determined using NMR spectroscopy, with other proteins in the Protein Data Bank suggested that chromo domains may act as protein interaction motifs. After using two-hybrid methods to identify proteins that interact with HP1, we characterised several proteins using biochemical and structural methods. In collaboration with Alain Verreault at CR-UK and Bruce Stillman at the Cold Spring Harbor Laboratory, we showed that shadow chromo domain of HP1 interacts with a chromatin assembly factor CAF-1. Using NMR spectroscopy, we have since determined the structures of the shadow chromo domain/CAF-1 complex, as well as that of the chromo domain/Lys-9 methylated histone H3 complex.

Based on these structures, we have constructed site-directed mutants that specifically knock out these interactions and have used these to study the function of HP1 further using molecular and cell biological methods.

For more details see: Nielsen et al., Nature, 2002, 416, 103-107 and Thiru et al., EMBO. J., 2004, 23, 489-99.

Our current research is focussed on studies of two key protein complexes involved in chromatin structure; Chromatin Assembly Factor-1 (CAF-1), which assembles histones H3/H4 into DNA in the first step of nucleosome assembly, and the Nucleosome Remodelling Deacetylase complex (NuRD), which shares two subunits RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively). RbAp46 and RbAp48 are highly homologous histone chaperones, which belong to the WD-40-repeat protein family and they are found in a large number of different complexes that either covalently modify histones or aid their assembly into nucleosomes.

We recently reported the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β-propeller structure and binds histone H4 in a groove formed between an N-terminal α-helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in a complex with ASF1, another histone chaperone. Our structure suggested that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix-1 of histone H4 unfolds to interact with the histone chaperone. We are currently employing a range of biochemical, biophysical and structural studies to investigate this hypothesis.

For more details see: Murzina et al., Structure, 2008, 16(7), 1077-1085

This work is supported by the Wellcome Trust. We are collaborating on this project also with Tony Carr at the University of Sussex, and with Tony Kouzarides at the Gurdon Institute in Cambridge.