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Biomedical subjects

P D Gregory

Publications and source records attributed to P D Gregory.

13 recordsLinked to original sources
PubMed2003

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

Beckwith-Wiedemann Syndrome↗
PubMed2001

Histone acetylation and chromatin remodeling.

Chromatin represents a repressive barrier to the process of transcription. This molecular obstacle is a highly dynamic structure, able to compact the DNA of the entire genome into the confines of a nucleus, and yet it allows access to the genetic information held within. The acetylation of histones has emerged as a regulatory mechanism capable of modulating the properties of chromatin and thus the competence of the DNA template for transcriptional activation. The role of acetylation in chromatin remodeling is therefore of paramount importance to our understanding of gene regulation in vivo.

Acetylation↗
PubMed2001

Transcription and chromatin converge: lessons from yeast genetics.

The control of transcription through the modification of chromatin has been a subject of intense study over the past year. The increasing use of genome-wide approaches to examine the role of chromatin and the complexes able to modify it is providing a global perspective that is profoundly altering our view of the transcription process.

Chromatin↗
PubMed2001

A transient histone hyperacetylation signal marks nucleosomes for remodeling at the PHO8 promoter in vivo.

Chromatin remodeling of the yeast PHO8 promoter requires the SAGA histone acetyltransferase complex. We report here that SAGA is necessary and sufficient to establish an activator-dependent hyperacetylation peak over the PHO8 promoter that is restricted to those nucleosomes that are remodeled upon activation. This local hyperacetylated state is observed upon activation in the absence of the SWI/SNF complex when the remodeling process is frozen subsequent to activator binding. Hyperacetylation is lost, however, if remodeling is permitted to go to completion. Thus, a transient histone hyperacetylation signal is shown to be a prerequisite for, and determinant of, the domain of nucleosome remodeling in vivo.

Acetylation↗
PubMed1999

Chromatin remodelling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding.

The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively. Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo. These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site. However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions. Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding. These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.

Acetyltransferases↗
PubMed1998

Chromatin and transcription--how transcription factors battle with a repressive chromatin environment.

The last year has seen much progress in our understanding of chromatin and transcription. Transcriptionally active chromatin has long been correlated with a higher level of histone acetlyation. The discovery of a nuclear histone acetyltransferase activity encoded by factors with a role in transcription raises the possibility that the cell is able to dynamically modulate the (local) level of histone acteylation, switching chromatin templates from inactive to transcriptionally active states. Furthermore, histone acetylation states have shown to play a role in determining the efficacy of transcriptionally silenced chromatin in both yeast and Drosophila. The advances in our knowledge regarding the role of the origin-recognition complex in the establishment of silencing, and the requirement for a locally concentrated zone of the silence information regulator proteins in the nucleus has provided insights into the complex architecture of silenced chromatin. The goal of understanding the mechanisms by which the cell is able to 'open' repressive chromatin structures has prompted the discovery of multiple chromatin remodelling activities. These large protein complexes identified from organisms as diverse as yeast, mouse, fly and man demonstrate the ubiquity and fundamental importance of the ability to perturb the structure of chromatin allowing transcription of the desired genes. These data provide the latest and potentially most significant demonstration of the importance of the nucleosome in the regulation of transcription.

Acetyltransferases↗
PubMed1998

Analyzing chromatin structure and transcription factor binding in yeast.

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.

Binding Sites↗
PubMed1998

Life with nucleosomes: chromatin remodelling in gene regulation.

In the past year, the role of chromatin has emerged at the forefront of transcription research. Discovery and characterisation of the chromatin modifying machinery have significantly advanced our understanding of the molecular activities that establish a transcriptionally competent substrate in vivo, and have underscored the importance of the part played by chromatin in the regulation of transcription.

Chromatin↗
PubMed1998

Absence of Gcn5 HAT activity defines a novel state in the opening of chromatin at the PHO5 promoter in yeast.

Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation. However, no effect on chromatin has been demonstrated. We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter. Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity. Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure. This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5. Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.

Acetyltransferases↗
PubMed1997

Studies of the repressor (BlaI) of beta-lactamase synthesis in Staphylococcus aureus.

Purified BlaI, the putative repressor of the beta-lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ-blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus, providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N- and C-termini. An operator DNA-protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the beta-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1-dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the beta-lactamase system.

Bacterial Proteins↗
PubMed1996

A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline.

The naturally occurring plasmid pSTS7 from Staphylococcus epidermidis mediated resistance to tetracycline via a tetL gene and to kanamycin and neomycin via an aadD gene. Plasmid pSTS7 showed partial restriction map and sequence homology to the previously described tetracycline resistance plasmid pNS1981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid pUB110 from S. aureus. Sequence analysis of the regions flanking the two resistance genes in pSTS7 led to the identification of a novel site for interplasmid recombination which could explain the derivation of pSTS7 from the incompatible pNS1981- and pUB110-like parental plasmids under tetracycline-selective pressure.

Amino Acid Sequence↗