PubMed Health⌕ Search

Biomedical subjects

Robert E Kingston

Publications and source records attributed to Robert E Kingston.

At least 19 recordsLinked to original sources
PubMed2006

Human ACF1 alters the remodeling strategy of SNF2h.

The human ACF chromatin-remodeling complex (hACF) contains the ATPase motor protein SNF2h and the non-catalytic hACF1 subunit. Here, we have compared the ability of SNF2h and a reconstituted hACF complex containing both SNF2h and hACF1 to remodel a series of nucleosomes containing different lengths of DNA overhang. Both SNF2h and hACF functioned in a manner consistent with sliding a canonical nucleosome. However, the non-catalytic subunit, hACF1, altered the remodeling properties of SNF2h by changing the nature of the requirement for a DNA overhang in the nucleosomal substrate and altering the DNA accessibility profile of the remodeled products. Surprisingly, addition of hACF1 to SNF2h increased the amount of DNA overhang needed to observe measurable amounts of DNA accessibility, but decreased the amount of overhang needed for a measurable binding interaction. We propose that these hACF1 functions might contribute to making the hACF complex more efficient at nucleosome spacing compared with SNF2h. In contrast, the SWI/SNF complex and its ATPase subunit BRG1 generated DNA accessibility profiles that were similar to each other, but different significantly from those of hACF and SNF2h. Thus, we observed divergent remodeling behaviors in these two remodeling families and found that the manner in which hACF1 alters the remodeling behavior of the ATPase is not shared by SWI/SNF subunits.

Adenosine Triphosphatases↗
PubMed2006

Characterization of the piRNA complex from rat testes.

Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (< or = 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex (piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals.

Adenosine Triphosphatases↗
PubMed2005

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the beta-globin locus control region.

Locus control regions (LCRs) are regulatory DNA sequences that are situated many kilobases away from their cognate promoters. LCRs protect transgenes from position effect variegation and heterochromatinization and also promote copy-number dependence of the levels of transgene expression. In this work, we describe the biochemical purification of a previously undescribed LCR-associated remodeling complex (LARC) that consists of heterogeneous nuclear ribonucleoprotein C1/C2, nucleosome remodeling SWI/SNF, and nucleosome remodeling and deacetylating (NuRD)/MeCP1 as a single homogeneous complex. LARC binds to the hypersensitive 2 (HS2)-Maf recognition element (MARE) DNA in a sequence-specific manner and remodels nucleosomes. Heterogeneous nuclear ribonucleoprotein C1/C2, previously known as a general RNA binding protein, provides a sequence-specific DNA recognition element for LARC, and the LARC DNA-recognition sequence is essential for the enhancement of transcription by HS2. Independently of the initiation of transcription, LARC becomes associated with beta-like globin promoters.

Amino Acid Sequence↗
PubMed2005

Swapping function of two chromatin remodeling complexes.

SWI/SNF- and ISWI-based complexes have distinct yet overlapping chromatin-remodeling activities in vitro and perform different roles in vivo. This leads to the hypothesis that the distinct remodeling functions of these complexes are specifically required for distinct biological tasks. By creating and characterizing chimeric proteins of BRG1 and SNF2h, the motor proteins of human SWI/SNF- and ISWI-based complexes, respectively, we found that a region that includes the ATPase domain specifies the outcome of the remodeling reaction in vitro. A chimeric protein based on BRG1 but containing the SNF2h ATPase domain formed an intact SWI/SNF complex that remodeled like SNF2h. This altered-function complex was active for remodeling and could stimulate expression from some, but not all, SWI/SNF responsive promoters in vivo. Thus, we were able to separate domains of BRG1 responsible for function from those responsible for SWI/SNF complex formation and demonstrate that remodeling functions are not interchangeable in vivo.

Adenosine Triphosphatases↗
PubMed2005

Analysis of a polycomb group protein defines regions that link repressive activity on nucleosomal templates to in vivo function.

Polycomb group (PcG) genes propagate patterns of transcriptional repression throughout development. The products of several such genes are part of Polycomb repressive complex 1 (PRC1), which inhibits chromatin remodeling and transcription in vitro. Genetic and biochemical studies suggest the product of the Posterior sex combs (Psc) gene plays a central role in both PcG-mediated gene repression in vivo and PRC1 activity in vitro. To dissect the relationship between the in vivo and in vitro activities of Psc, we identified the lesions associated with 11 genetically characterized Psc mutations and asked how the corresponding mutant proteins affect Psc activity on nucleosomal templates in vitro. Analysis of both single-mutant Psc proteins and recombinant complexes containing mutant protein revealed that Psc encodes at least two functions, complex formation and the inhibition of remodeling and transcription, which require different regions of the protein. There is an excellent correlation between the in vivo phenotypes of mutant Psc alleles and the structure and in vitro activities of the corresponding proteins, suggesting that the in vitro activities of PRC1 reflect essential functions of Psc in vivo.

Animals↗
PubMed2004

Chromatin compaction by a polycomb group protein complex.

Polycomb group proteins preserve body patterning through development by maintaining transcriptional silencing of homeotic genes. A long-standing hypothesis is that silencing involves creating chromatin structure that is repressive to gene transcription. We demonstrate by electron microscopy that core components of Polycomb Repressive Complex 1 induce compaction of defined nucleosomal arrays. Compaction by Polycomb proteins requires nucleosomes but not histone tails. Each Polycomb complex can compact about three nucleosomes. A region of Posterior Sex Combs that is important for gene silencing in vivo is also important for chromatin compaction, linking the two activities. This mechanism of chromatin compaction might be central to stable gene silencing by the Polycomb group.

Chromatin↗
PubMed2004

Propagation of silencing; recruitment and repression of naive chromatin in trans by polycomb repressed chromatin.

The Polycomb group (PcG) proteins maintain stable and heritable repression of homeotic genes. Typically, Polycomb response elements (PRE) that direct PcG repression are located at great distances (10s of kb) from the promoters of PcG-repressed genes, and it is not known how these PREs can communicate with promoters over such distances. Using Class II mouse PRC core complexes (mPCCs) assembled from recombinant subunits, we investigated how PcG complexes might bridge distant chromosomal regions. Like native and recombinant Drosophila Class II complexes, mPCC represses chromatin remodeling and transcription. Interestingly, mPCC bound to one polynucleosome template can recruit a second template from solution and renders it refractory to transcription and chromatin remodeling. A Drosophila PRC core complex (dPCC) also is able to recruit a second template. Posterior sex combs (PSC), a subunit of dPCC, inhibits chromatin remodeling and transcription efficiently but requires assembly with dRING1 to recruit chromatin. Thus, repression and template bridging require different subunits of PcG complexes, suggesting that long-range effects may be mechanistically distinct from repression.

Animals↗
PubMed2004

Division of labor in polycomb group repression.

The epigenetic maintenance of gene expression patterns is essential for developing and maintaining the diverse types of cell that cooperate to form the larger organism. Recent data suggest that proteins of the Polycomb group (PcG) use a combination of posttranslational modifications and structural changes to the underlying chromatin structure to maintain silenced epigenetic states. We are now beginning to understand the mechanisms by which the PcG proteins are able to silence genes and to maintain this silencing over many cell divisions.

Animals↗
PubMed2004

Transcriptional regulation and binding of heat shock factor 1 and heat shock factor 2 to 32 human heat shock genes during thermal stress and differentiation.

Transcription of mammalian heat shock genes can be regulated by heat shock factors (HSF) 1 and 2. Although it has been shown previously that these factors respond to distinct stimuli, a broad analysis of the induction and function of these factors in living cells has not been performed. In our study, we assayed binding of human HSF1 and HSF2 at the promoters of 32 genes identified through LocusLink as heat shock genes in response to elevated temperature and hemin-induced differentiation in human K562 erythroleukemic cells using the chromatin immunoprecipitation technique. We also measured the induced expression of these genes under these 2 conditions. We found that 17 of the 32 genes were transcriptionally induced during heat shock, and HSF1 binding was detected at 15 of the 17 promoters. Nearly all the genes induced by heat shock were also induced to a lesser degree during hemin treatment. However, some genes were induced significantly more during hemin treatment than during heat shock. A new finding is that HSF1 and HSF2 bind to the same targets, but HSF1 binding is activated more by heat than by hemin treatment, and HSF2 binding is only activated by hemin treatment and not by heat. This technology also identified previously unknown HSF1 binding sites near genes that were previously shown to be heat inducible that may contribute to gene-specific regulation.

Base Sequence↗
PubMed2004

Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

Amino Acid Sequence↗
PubMed2003

Regulation of Polycomb group complexes by the sequence-specific DNA binding proteins Zeste and GAGA.

Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. We have used purified proteins and complexes reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1.

Animals↗
PubMed2003

Elevated expression of heat shock factor (HSF) 2A stimulates HSF1-induced transcription during stress.

Heat shock factor 2 (HSF2) belongs to a family of structurally related transcription factors, which share the property of binding to heat shock elements in the promoters of hsp molecular chaperone genes. However, unlike HSF1, which is essential for hsp gene transcription, the cellular functions of HSF2 are not well known. Here we show that human HSF2, although an ineffective activator of the hsp70 promoter in vitro and in vivo in the absence of stress, participates in the activation of the hsp70 promoter by heat shock. HSF2 was not, however, activated by heat shock in cells deficient in functional HSF1, suggesting a requirement for HSF1 in HSF2-mediated transcriptional enhancement. In addition, HSF2 regulation involves differential activity of two isoforms, HSF2A and HSF2B, which arise from alternative splicing of a common hsf2 gene. Under basal conditions, both HSF2 isoforms are ineffective in activating the hsp70 transcription. However, heat shock differentially activates HSF2A in vivo. This phenomenon appears to be physiologically significant, as human myeloprogenitor cells differentiating along the erythroid lineage express HSF2A de novo and undergo a large increase in capacity to activate the hsp70 promoter. Our experiments further show that HSF1 is physically associated with HSF2 in the cell and that such binding is enhanced by heat shock. Our data suggest a mechanism involving the formation of heterocomplexes between HSF1 and HSF2 with enhanced activity to activate the hsp70 promoter when compared with HSF1 or HSF2 homotrimers.

Amino Acid Sequence↗
PubMed2003

Localized recruitment of a chromatin-remodeling activity by an activator in vivo drives transcriptional elongation.

To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mouse hsp70 gene. A mutation that diminished transcriptional initiation led to twofold reductions in hsp70 mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from the hsp70 promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-length hsp70 mRNA.

Animals↗
PubMed2003

Distinct strategies to make nucleosomal DNA accessible.

One hallmark of ATP-dependent remodeling complexes is the ability to make nucleosomal DNA accessible to regulatory factors. We have compared two prominent human ATP-dependent remodelers, BRG1 from the SWI/SNF family and SNF2h from the ISWI family, for their abilities to make a spectrum of nucleosomal sites accessible. By measuring rates of remodeling at seven different sites on a mononucleosome and at six different sites on the central nucleosome of a trinucleosome, we have found that BRG1 opens centrally located sites more than an order of magnitude better than SNF2h. We provide evidence that this capability of BRG1 is caused by its ability to create DNA loops on the surface of a nucleosome, even when that nucleosome is constrained by adjacent nucleosomes. This specialized ability to make central sites accessible should allow SWI/SNF family complexes to facilitate binding of nuclear factors in chromatin environments where adjacent nucleosomes might otherwise constrain mobility.

Adenosine Triphosphate↗
PubMed2002

Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.

Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (ESC) and Enhancer of Zeste [E(Z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(Z) suggests this complex might methylate histones. We purified an ESC-E(Z) complex from Drosophila embryos and found four major subunits: ESC, E(Z), NURF-55, and the PcG repressor, SU(Z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(Z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing.

Animals↗
PubMed2002

Cooperation between complexes that regulate chromatin structure and transcription.

Chromatin structure creates barriers for each step in eukaryotic transcription. Here we discuss how the activities of two major classes of chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes, might be coordinated to create a DNA template that is accessible to the general transcription apparatus.

Acetyltransferases↗
PubMed2002

Effect of delays in primary care referral on survival of women with epithelial ovarian cancer: retrospective audit.

OBJECTIVE: To examine referral pathways from primary care for patients with epithelial ovarian cancer and to identify factors related to survival at 18 months. DESIGN: Retrospective review of patient notes. SETTING: General practices and receiving hospitals within Mersey region. SUBJECTS: 135 patients with epithelial ovarian cancer identified from an audit in the Mersey area between 1992 and 1994. MAIN OUTCOME MEASURES: Delays between onset of symptoms and treatment attributable to patient, general practitioner, and hospital. RESULTS: 105 (78%) women first presented to their general practitioner within four weeks of the onset of symptoms. 99 (73%) women were referred to hospital by their general practitioners within four weeks of presentation, and 95 (70%) were seen in hospital within two weeks of referral. Multivariate analysis with survival as the dependent variable identified age (odds ratio 0.96, 95% confidence interval 0.93 to 0.99) cancer stage III or more (0.15, 0.05 to 0.43), and non-specific symptoms (0.36, 0.14 to 0.89) as significant variables. CONCLUSION: Most patients attended their general practitioner within four weeks and were referred within two weeks. No evidence was found that delays in referral or diagnosis adversely affected survival at 18 months. Stage of disease at surgery was the most important adverse factor. An effective screening programme is the most likely method to improve survival.

Aged↗