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Biomedical subjects

Christopher J Fry

Publications and source records attributed to Christopher J Fry.

4 recordsLinked to original sources
PubMed2006

The LRS and SIN domains: two structurally equivalent but functionally distinct nucleosomal surfaces required for transcriptional silencing.

Genetic experiments have identified two structurally similar nucleosomal domains, SIN and LRS, required for transcriptional repression at genes regulated by the SWI/SNF chromatin remodeling complex or for heterochromatic gene silencing, respectively. Each of these domains consists of histone H3 and H4 L1 and L2 loops that form a DNA-binding surface at either superhelical location (SHL) +/-2.5 (LRS) or SHL +/-0.5 (SIN). Here we show that alterations in the LRS domain do not result in Sin(-) phenotypes, nor does disruption of the SIN domain lead to loss of ribosomal DNA heterochromatic gene silencing (Lrs(-) phenotype). Furthermore, whereas disruption of the SIN domain eliminates intramolecular folding of nucleosomal arrays in vitro, alterations in the LRS domain have no effect on chromatin folding in vitro. In contrast to these dissimilarities, we find that the SIN and LRS domains are both required for recruitment of Sir2p and Sir4p to telomeric and silent mating type loci, suggesting that both surfaces can contribute to heterochromatin formation. Our study shows that structurally similar nucleosomal surfaces provide distinct functionalities in vivo and in vitro.

Alleles↗
PubMed2003

A native peptide ligation strategy for deciphering nucleosomal histone modifications.

Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies. However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays. This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications.

Acetyltransferases↗
PubMed2002

Modulating acetyl-CoA binding in the GCN5 family of histone acetyltransferases.

The histone acetyltransferase (HAT) GCN5 is the founding member for a family of chromatin remodeling enzymes. GCN5 is the catalytic subunit of a large multi-subunit complex that functions in the regulation of gene activation via acetylation of lysine residues within the N-terminal tails of core histone proteins. Using acetyl-CoA as a co-substrate, the high affinity binding of acetyl-CoA is a critical first step in the reaction. Here, we examine the biochemical and biological importance of a conserved hydroxyl-bearing residue in signature motif A. Interestingly, one major exception is the Saccharomyces cerevisiae GCN5, where an alanine (Ala(190)) is located in the corresponding position. In related GCN5 family structures, a hydroxyl-containing side chain residue is hydrogen-bonded to the alpha-phosphate oxygen of CoA. We demonstrate that this key hydrogen bond contributes approximately 10-fold to the binding affinity of GCN5 HATs for acetyl-CoA. Human p300/CBP-associating factor, human GCN5, and tetrahymena GCN5 displayed dissociation constants (K(d)) for acetyl-CoA of 0.64 +/- 0.12, 0.56 +/- 0.15, and 0.62 +/- 0.17 microm, respectively. In contrast, S. cerevisiae GCN5 displayed a K(d) of 8.5 microm. When Ala(190) was replaced with threonine, the A190T derivative yielded a K(d) value of 0.56 +/- 0.1 microm for acetyl-CoA, completely restoring the higher affinity binding seen with the GCN5 homologs that naturally harbor a threonine at this position. Detailed kinetic analyses revealed that the A190T derivative was otherwise catalytically indistinguishable from wild type GCN5. We also demonstrate that the A190T allele rescued the slow growth phenotype and the defect in HO transcription caused by a deletion of GCN5. Furthermore, the A190T allele supported wild type levels of transcriptionally targeted and global histone H3 acetylation. In each case, the A190T derivative behaved similarly to wild type GCN5, suggesting that the efficacy of HAT activity by GCN5 is not limited by the availability of nuclear acetyl-CoA pools.

Acetyl Coenzyme A↗