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D Bertwistle

Publications and source records attributed to D Bertwistle.

14 recordsLinked to original sources
PubMed2002

BRCA1 and BRCA2 bind Stat5a and suppress its transcriptional activity.

Germline mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are thought to account for a large portion of familial breast cancer. The increased risk of breast cancer in women carrying such mutations suggests that these proteins play a critical role in the growth regulation of mammary epithelial cells. Another protein, Stat5a, is known to be essential for growth and terminal differentiation of breast epithelial cells. Here we show that Stat5a forms a complex with both BRCA1 and BRCA2 in breast epithelial cells upon stimulation with prolactin. In addition, we show that the activity of Stat5a on the beta-casein promoter is modulated by both BRCA1 and BRCA2. This interaction may be important during the expansion and terminal differentiation of breast epithelial cells, as happens during pregnancy and lactation.

Animals↗
PubMed2001

Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences.

Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology- directed double-strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error-prone repair by homology-directed single-strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error-prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C-induced DNA cross-links at the expense of error-free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2.

Alleles↗
PubMed2000

BRCA1 and BRCA2.

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BRCA1 Protein↗
PubMed1999

The pathology of familial breast cancer: How do the functions of BRCA1 and BRCA2 relate to breast tumour pathology?

Women with mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, have an increased risk of developing breast cancer. Both BRCA1 and BRCA2 are thought to be tumour suppressor genes since the wild type alleles of these genes are lost in tumours from heterozygous carriers. Several functions have been proposed for the proteins encoded by these genes which could explain their roles in tumour suppression. Both BRCA1 and BRCA2 have been suggested to have a role in transcriptional regulation and several potential BRCA1 target genes have been identified. The nature of these genes suggests that loss of BRCA1 could lead to inappropriate proliferation, consistent with the high mitotic grade of BRCA1-associated tumours. BRCA1 and BRCA2 have also been implicated in DNA repair and regulation of centrosome number. Loss of either of these functions would be expected to lead to chromosomal instability, which is observed in BRCA1 and BRCA2-associated tumours. Taken together, these studies give an insight into the pathogenesis of BRCA-associated tumours and will inform future therapeutic strategies.

BRCA1 Protein↗
PubMed1999

Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification.

Women heterozygous for mutations in the breast-cancer susceptibility genes BRCA1 and BRCA2 have a highly elevated risk of developing breast cancer [1]. BRCA1 and BRCA2 encode large proteins with no sequence similarity to one another. Although involvement in DNA repair and transcription has been suggested, it is still not understood how loss of function of these genes leads to breast cancer [2]. Embryonic fibroblasts (MEFs) derived from mice homozygous for a hypomorphic mutation (Brca2(Tr2014)) within the 3' region of exon 11 in Brca2 [3], or a similar mutation (Brca2(Tr)) [4], proliferate poorly in culture and overexpress the tumour suppressor p53 and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). These MEFs have intact p53-dependent DNA damage G(1)-S [3] [4] and G(2)-M checkpoints [4], but are impaired in DNA double-strand break repair [3] and develop chromosome aberrations [4]. Here, we report that Brca2(Tr2014/Tr2014) MEFs frequently develop micronuclei. These abnormal DNA-containing bodies were formed through both loss of acentric chromosome fragments and by chromosome missegregation, which resulted in aneuploidy. Absence of Brca2 also led to centrosome amplification, which we found associated with the formation of micronuclei. These data suggest a potential mechanism whereby loss of BRCA2 may, within subclones, drive the loss of cell-cycle regulation genes, enabling proliferation and tumourigenesis.

Aneuploidy↗
PubMed1999

Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.

Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.

Amino Acid Sequence↗
PubMed1998

Functions of the BRCA1 and BRCA2 genes.

Mutations in the BRCA1 and BRCA2 genes confer a high risk of breast cancer development. Both genes encode very large proteins of unknown function but recent results suggest that they may have roles in transcriptional regulation and DNA repair. These advances offer the prospect of understanding not only the normal cellular function of these genes but also how their loss leads to tumour formation.

Animals↗
PubMed1997

Nuclear location and cell cycle regulation of the BRCA2 protein.

Women carrying a germ-line mutation in the BRCA1 or BRCA2 genes have a high risk of developing breast cancer, and loss of the wild-type allele in tumors suggests that these genes function as tumor suppressor genes. The BRCA2 gene encodes a 3418-amino acid protein with no significant sequence similarity to any known protein. To begin to elucidate the cellular role of BRCA2, we have raised antibodies to the BRCA2 protein and used these to study its subcellular localization and expression. We show that BRCA2 is a nuclear protein expressed in response to cell proliferation and that BRCA2 expression is initiated before DNA synthesis.

Animals↗
PubMed1997

GATA-2 is a maternal transcription factor present in Xenopus oocytes as a nuclear complex which is maintained throughout early development.

We show that Xenopus oocytes and embryos contain GATA-2, stored in nuclei as a non-chromatin-bound complex. Its binding site specificity is different from that of GATA-1, having a much higher affinity for the motif with a core GATC sequence. This binding site preference was markedly reduced by either release of the factor with deoxycholate or purification on a DNA affinity column, suggesting a role for a cofactor(s). The identity of the maternal GATA factor was established as GATA-2 in two ways: (1) binding to an oligonucleotide probe was abolished by inclusion of either of two GATA-2 monoclonal antibodies, and (2) a protein of correct molecular weight for GATA-2 was detected by immunoblotting with a polyclonal antibody raised against a Xenopus GATA-2-specific peptide. Although predominantly complexed, some of the oocyte GATA-2 is functional as a transcription factor because the transcriptional activity of the chicken betaH-globin promoter injected into oocytes was reduced by mutation of either of two GATA binding sites. This effect was more pronounced when the stronger of the two sites was mutated. Butyrate treatment of oocytes stimulated cap-site initiation by up to 17-fold with both normal promoter and GATA site mutant constructs, showing that the mechanism of butyrate stimulation is not via GATA-2. The possible significance of regulating the availability of maternal GATA-2 during early development is discussed.

Animals↗
PubMed1997

Over-expression of GATA-6 in Xenopus embryos blocks differentiation of heart precursors.

Xenopus GATA-6 transcripts are first detected at the beginning of gastrulation in the mesoderm, and subsequent domains of expression include the field of cells shown to have heart-forming potential. In this region, GATA-6 expression continues only in those cells that go on to form the heart; however, a decrease occurs prior to terminal differentiation. Artificial elevation of GATA-6, but not GATA-1, prevents expression of both cardiac actin and heart-specific myosin light chain. This effect is heart-specific because cardiac actin expression is unaffected in somites. Expression of the earlier marker XNkx-2.5 was unaffected and morphological development of the heart was initiated independently of the establishment of the contractile machinery. We conclude that a reduction in the level of GATA-6 is important for the progression of the cardiomyogenic differentiation programme and that GATA-6 may act to maintain heart cells in the precursor state. At later stages, when the elevated GATA-6 levels had decayed, differentiation ensued but the number of cells contributing to the myocardium had increased, suggesting either that the blocked cells had proliferated or that additional cells had been recruited.

Alleles↗
PubMed1997

Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation.

Germline mutation of the BRCA2 gene carries a high risk of developing breast cancer. To study the function of this gene, we generated a mutation in Brca2 in mice. Unlike other mutations in the Brca2 gene, which are lethal early in embryogenesis when homozygous, some of our homozygous mutant mice survive to adulthood. These animals have a wide range of defects, including small size, improper differentiation of tissues, absence of germ cells and the development of lethal thymic lymphomas. Fibroblasts cultured from BrcaZ-/-embryos have a defect in proliferation that may be mediated by over-expression of p53 and p21Waf1/CIP1. We show that Brca2 is required for efficient DNA repair, and our results suggest that loss of the p53 checkpoint may be essential for tumour progression triggered by mutations in BRCA2.

Animals↗
PubMed1996

GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid.

The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP. The VBI contributes to both embryonic and adult blood, whereas the DLP contains precursors of adult blood only, which have not been identified previously with molecular markers. The possibility that the GATA-2-expressing cells between the VBI and DLP were haematopoietic progenitors migrating from the VBI to the DLP was ruled out by transplantation analysis. Differential effects of RA on the expression of GATA-1 and GATA-2 suggest that RA has a direct action on haematopoietic differentiation, rather than on the formation of haematopoietic mesoderm.

Animals↗
PubMed1994

Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands.

To increase our understanding of haematopoiesis during early vertebrate development, we have studied the expression pattern of the transcription factor GATA-2 in Xenopus embryos, and asked how this is regulated. We show that the blood island precursors of the ventral mesoderm express GATA-2 RNA at neural tube stages, some 5 hours before globin RNA is detected in their derivatives. Prior to this however, GATA-2 is expressed much more widely within the embryo. Maternal transcripts are uniformly distributed, and zygotic transcription is activated during gastrulation throughout ventral and lateral regions of the embryo, with expression highest in the sensorial ectoderm and only weak in the ventral mesoderm. The domain of GATA-2 expression in neurulae outlines the region of the neural plate and suggests a possible wider role in dorsoventral patterning. To identify the signals involved in regulating this pattern of expression, we performed experiments with embryo explants. GATA-2 is activated autonomously in isolated animal caps and this activation is suppressed by the mesoderm-inducing factor activin, but not by FGF. Thus, the down-regulation of GATA-2 observed in the region of the Spemann organiser may be a response to an activin-like signal emanating from the dorsal-vegetal region or Nieuwkoop centre. GATA-2 activation in animal caps and ventral marginal zones was suppressed by co-culturing with dorsal marginal zones, suggesting that a signal from the Spemann organiser is involved in suppression of GATA-2 in the dorsal region of the embryo. Expression of a candidate for this signal, noggin, had the same effect. Taken together, the observations presented here suggest that GATA-2 activation occurs by default in the absence of signals, that the restriction of its expression within the early embryo is controlled by negative signals emanating from the Nieuwkoop centre and the organiser, and that noggin and activin-like molecules play a role in these signalling pathways.

Activins↗