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I G Cowell

Publications and source records attributed to I G Cowell.

At least 19 recordsLinked to original sources
PubMed2001

The gene and pseudogenes of Cbx3/mHP1 gamma.

The HP1 class of chromobox (Cbx) genes encode an evolutionarily conserved family of proteins involved in the packaging of chromosomal domains into a repressive heterochromatic state. The murine Cbx5, Cbx1 and Cbx3 genes encode the three mouse HP1 proteins, mHP1 alpha, -beta and -gamma respectively. Here, we report the cloning of the mouse Cbx3/HP1 gamma gene and the chromosomal localisation of Cbx3 and three Cbx3-related pseudogenes. The Cbx3 structural gene is located on mouse Chromosome 6, close to the Hoxa cluster. Two Cbx3 processed pseudogenes are separated by just 300 bp and are arranged in a head-to-tail configuration on Chromosome 13 while a third pseudogene is found on mouse Chromosome 4. The genomic intron-exon arrangement of Cbx3 is different from the conserved organisation of three other mammalian HP1 genes, Cbx1 (mHP1 beta), CBX3 (hHP1 gamma), and Cbx5 (mHP1 alpha) in that Cbx3 lacks an intron that is present in the others.

Amino Acid Sequence↗
PubMed2000

Dynamic associations of heterochromatin protein 1 with the nuclear envelope.

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to purified nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His(6)-tagged peptide representing this region inhibited recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.

Acetylation↗
PubMed2000

Human topoisomerase IIalpha and IIbeta interact with the C-terminal region of p53.

The p53 tumor suppressor protein is a critical regulator of cell cycle progression and apoptosis following exposure of cells to DNA damaging agents such as ionizing radiation or anticancer drugs. An important group of anticancer drugs, including compounds such as etoposide and doxorubicin (Adriamycin), interacts with DNA topoisomerase II (topo II), causing the accumulation of enzyme-DNA adducts that ultimately lead to double-strand breaks and cell death via apoptosis. Human topo IIbeta has previously been shown to interact with p53, and we have extended this analysis to show that both topo IIalpha and IIbeta interact with p53 in vivo and in vitro. Furthermore, we show that the regulatory C-terminal basic region of p53 (residues 364-393) is necessary and sufficient for interaction with DNA topo II.

Alternative Splicing↗
PubMed2000

Mammalian chromodomain proteins: their role in genome organisation and expression.

The chromodomain is a highly conserved sequence motif that has been identified in a variety of animal and plant species. In mammals, chromodomain proteins appear to be either structural components of large macromolecular chromatin complexes or proteins involved in remodelling chromatin structure. Recent work has suggested that apart from a role in regulating gene activity, chromodomain proteins may also play roles in genome organisation. This article reviews progress made in characterising mammalian chromodomain proteins and emphasises their emerging role in the regulation of gene expression and genome organisation. BioEssays 22:124-137, 2000.

Amino Acid Sequence↗
PubMed2000

Conservation of heterochromatin protein 1 function.

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.

Amino Acid Sequence↗
PubMed1998

Nuclear distribution of human DNA topoisomerase IIbeta: a nuclear targeting signal resides in the 116-residue C-terminal tail.

We have analyzed the subcellular distribution of the beta isoform of human topoisomerase II using both isoform-specific antisera and an epitope-tagging approach. Previous immunocytochemical studies have yielded differing results with one reporting this isoform to be predominantly nucleolar. Later studies seem to refute this finding, as do our results with isoform-specific antisera reported here. Epitope tagging minimizes potential complications arising from the use of anti-topoisomerase II antisera that may recognize epitopes that are modified or masked in vivo and could lead to misleading results in immunocytochemical studies. A second strength of this approach is that it allowed a comparison with similarly tagged control proteins (derived from the nucleolar transcription factor UBF) that were known to localize unambiguously to the cytoplasmic, nucleoplasmic, or nucleolar compartments. We report that the C-terminal domain of topoisomerase IIbeta fused to a beta-galactosidase tag localizes to the nucleus (but not the nucleolar compartment) and that this is indistinguishable from the localization of native topoisomerase IIbeta detected by isoform-specific antisera. Further analysis revealed that the nuclear localization determinant lies within the 116-residue C-terminal tail of human topoisomerase IIbeta.

Amino Acid Sequence↗
PubMed1997

Self-association of chromo domain peptides.

The chromo domain is a sequence motif first recognised in the Drosophila polycomb protein and heterochromatin protein (HP1), two proteins associated with stable and heritable transcriptional silencing. Polycomb is one of a number of genes that are required to prevent ectopic homeotic gene expression in Drosophila, while HP1, the product of the Drosophila melanogaster Su(var)205 gene, is associated with the phenomenon of position effect variegation. These proteins are believed to be components of chromatin-associated multi-protein complexes that bring about stable transcriptional silencing and the chromo domain has been implicated in chromatin targeting, probably through protein-protein interaction. Recently, mammalian homologues of both polycomb and HP1 have been described. Here we demonstrate for the first time that oligopeptides containing a chromo domain derived from the mouse polycomb homologue M33 form multimeric complexes in solution, supporting the role of the chromo domain in multiprotein complex assembly.

Animals↗
PubMed1996

Protein-protein interaction between the transcriptional repressor E4BP4 and the TBP-binding protein Dr1.

We have previously mapped a repression domain from the active transcriptional repressor E4BP4 to a 65 amino acid segment near the C-terminus of the polypeptide. Here we show that the E4BP4 repression domain interacts specifically with the TBP binding repressor protein Dr1. Mutants that affect the ability of E4BP4 to bring about transcriptional repression are also deficient in their binding of Dr1. The results are discussed in the light of evidence for squelching of a 'global' repressor by a DNA binding defective E4BP4 mutant.

Adenovirus E1A Proteins↗
PubMed1994

Transcriptional repression by the human bZIP factor E4BP4: definition of a minimal repression domain.

The bZIP factor E4BP4 overlaps in DNA binding site specificity with the transcriptional activator CREB and members of the ATF family of transcription factors, but is an active transcriptional repressor. In this study we have mapped the repressing activity of E4BP4 to a small 'domain' of 65 amino acids that retains its ability to repress transcription when transferred to the heterologous DNA binding domain of the yeast transcriptional activator GAL4. This segment of the E4BP4 polypeptide contains a high proportion of charged amino acids and does not resemble the repression domains that have been characterized so far from other active transcriptional repressors such as the Drosophila Krüppel, Engrailed or Even-skipped proteins. A mutation which changes the charge configuration of this repression module resulted in a complete loss of repressor activity. The E4BP4-GAL4 fusion protein is able to repress the residual transcription from minimal promoters containing the adenovirus E4 or E1b TATA box. This is consistent with a mechanism of action whereby E4BP4 interacts with some component of the general transcription machinery to cause repression of basal and activated transcription. Although a number of nuclear proteins are able to interact with the E4BP4 repression domain in vitro, these proteins do not appear to include the general transcription factors TFIIB or TBP.

Amino Acid Sequence↗
PubMed1994

Repression versus activation in the control of gene transcription.

Studies on the regulation of transcription often focus on mechanisms of transcriptional activation. However, transcriptional repression is also an important factor in the regulation of many genes. Transcription of specific genes can be downregulated in various ways, and examination of a number of different systems has revealed that most or all steps required for transcriptional activation can be interfered with by transcriptional repressors.

Amino Acid Sequence↗
PubMed1992

Transcriptional repression by a novel member of the bZIP family of transcription factors.

We describe here a novel member of the bZIP family of DNA-binding proteins, designated E4BP4, that displays an unusual DNA-binding specificity which overlaps that of the activating transcription factor family of factors. When expressed in a transient transfection assay with a suitable reporter plasmid, E4BP4 strongly repressed transcription in a DNA-binding-site-dependent manner. Examination of a series of deletion mutants revealed that sequences responsible for the repressing potential of E4BP4 lie within the carboxyl-terminal region of the protein. No similarity was found between this region and the repressing domains of other known eukaryotic transcriptional repressors.

Amino Acid Sequence↗
PubMed1991

Glutathione transferase pi its minimal promoter and downstream cis-acting element.

Fragments of the human glutathione S-transferase pi gene and 15 kb of its 5' flanking region have been fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Transfection into a number of human cell lines (Hela, HepG2, MCF7 and EJ) has demonstrated that the AP1 binding site, located between nucleotides -58 and -65 (Cowell et al. 1988. Biochem. J. 255, 79-83), is essential for basal level promoter activity. We have also identified a positive cis-acting DNA element between nucleotides +8 and +72 which seems to be part of the promoter. No other regulatory activity was identified within the 17 kb analyzed.

Animals↗
PubMed1989

Control of expression of the human glutathione S-transferase pi gene differs from its rat orthologue.

We have examined regulation of the glutathione S-transferase pi gene by transient expression assay, and find that a fragment from 8 to 99 bp upstream of the cap site promotes transcription, but there is no evidence for any enhancer activity in a further 6 kb of flanking sequence. Analysis of this sequence by reference to a primate sequence database and Southern blotting revealed that as much as 5 kb of this flanking DNA were composed of repetitive insertion elements including an Alu and a LINE 1 repeat. The promoter fragment has been sequenced (Cowell et al (1988) Biochem. J. 255, 79-83) and contains a consensus AP1 binding site; in some cases, these have been associated with transcriptional induction by phorbol esters and ras oncogenes. We measured the steady state levels of glutathione S-transferase pi mRNA in human cell lines which were known to express ras oncogenes and compared them to human cell lines which have not been identified with ras activation. There was no correlation between expression of activated ras and expression of glutathione S-transferase pi mRNA. Treatment of HeLa cells, HepG2 cells and a small cell lung carcinoma line, GLC 8, with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate failed to alter the steady state levels of endogenous glutathione S-transferase pi mRNA. The differences between these results and those of similar studies on rat glutathione S-transferase subunit 7, a structural orthologue of glutathione S-transferase pi, are discussed.

Animals↗
PubMed1988

The structure of the human glutathione S-transferase pi gene.

The human glutathione S-transferase pi gene has been isolated from a cosmid library. The gene spans approximately 3 kb, is interrupted by six introns and the region around its 5' end has the high G + C and CpG content typical of an HTF (HpaII tiny fragment) island. In addition to a TATA box at position -28 relative to the transcription initiation site and two G + C boxes (GGGCGG), the promoter contains a sequence motif matching the phorbol ester- and ras-responsive element from the polyoma virus enhancer.

Amino Acid Sequence↗