BRCA1 transcriptionally regulates genes associated with the basal breast cancer phenotype

Background Ten to twenty per cent of breast tumours exhibit a basallike genetic profile and these tumours carry a poor prognosis. Breast tumours which contain germline mutations for BRCA1 commonly exhibit a molecular profile similar to basal breast tumours. BRCA1 is a tumour suppressor gene which is mutated in up to 5–10% of breast cancer cases and is involved in multiple cellular processes including DNA damage control, cell cycle checkpoint control, apoptosis, ubiquitination and transcriptional regulation.

Methods Microarray-based profiling was carried out using the HCC1937EV and HCC1937BR breast cancer cell lines. Basal gene and protein expression levels were analysed by qRT-PCR and western blotting. ChIP analyses were performed and demonstrated that BRCA1 regulates basal gene expression through a transcriptional mechanism involving c-myc.

Results We have previously carried out microarray-based expression profiling to examine differences in gene expression when BRCA1 is reconstituted in BRCA1 mutated HCC1937 breast cancer cells. We observed that p-cadherin and the cytokeratin 5 and cytokeratin 17 genes, which are strongly correlated with the basal phenotype, are differentially expressed when BRCA1 is reconstituted. In addition, qRT-PCR and ChIP analysis of BRCA1 reconstituted cells show that BRCA1 represses the expression of these basal genes by a transcriptional mechanism. Furthermore, abrogation of endogenous BRCA1 protein in the T47D cell line using siRNA results in reexpression of these basal genes, suggesting that BRCA1 expression levels may be important in basal gene expression. We have also demonstrated that BRCA1 is physically associated with the promoter regions of basal genes through an association with c-myc. Consequently, we have confirmed that siRNA inhibition of c-myc in T47D cells results in re-expression of these genes.

Conclusions Our results suggest that BRCA1 is involved in the transcriptional regulation of genes associated with the basal phenotype and that BRCA1 controls basal gene expression through a transcriptional mechanism involving c-myc. Further work is now concentrating on defining the relationship between BRCA1 and basal gene expression and how this may affect clinical responses to breast cancer chemotherapy.

Profiling of the BRCA1 transcriptome through microarray and ChIP-chip analysis

A role for BRCA1 in the direct and indirect regulation of transcription is well established. However, a comprehensive view of the degree to which BRCA1 impacts transcriptional regulation on a genome-wide level has not been defined. We performed genome-wide expression profiling and ChIP-chip analysis, comparison of which revealed that although BRCA1 depletion results in transcriptional changes in 1294 genes, only 44 of these are promoter bound by BRCA1. However, 27 of these transcripts were linked to transcriptional regulation possibly explaining the large number of indirect transcriptional changes observed by microarray analysis. We show that no specific consensus sequence exists for BRCA1 DNA binding but rather demonstrate the presence of a number of known and novel transcription factor (TF)- binding sites commonly found on BRCA1 bound promoters. Co-immunoprecipitations confirmed that BRCA1 interacts with a number of these TFs including AP2-a, PAX2 and ZF5. Finally, we show that BRCA1 is bound to a subset of promoters of genes that are not altered by BRCA1 loss, but are transcriptionally regulated in a BRCA1-dependent manner upon DNA damage. These data suggest a model, whereby BRCA1 is present on defined promoters as part of an inactive complex poised to respond to various genotoxic stimuli. © The Author(s) 2011. Published by Oxford University Press.

Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs). Using integrative strategies, we demonstrate that only a subset of these sites are bound by TP53 in response to DNA damage. We identify a role for TP63 in transcriptional regulation of multiple genes genetically linked to cleft palate and identify AP-2alpha (TFAP2A) as a co-regulator of a subset of these genes. We further demonstrate that AP-2gamma (TFAP2C) can bind a subset of these regions and that acute depletion of either TFAP2A or TFAP2C alone is sufficient to reduce terminal differentiation of organotypic epidermal skin equivalents, indicating overlapping physiological functions with TP63.

Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Deregulated estrogen metabolism in BRCA1 deficient cells induces chromosomal instability.

Objectives: Germline mutations in BRCA1 predispose carriers to a high
incidence of breast and ovarian cancers. The BRCA1 protein functions to maintain
genomic stability via important roles in DNA repair, transcriptional regulation, and
post-replicative repair. Despite functions in processes essential in all cells, BRCA1
loss or mutation leads to tumours predominantly in estrogen-regulated tissues.
Here, we aim to determine if endogenous estrogen metabolites may be an initiator
of genomic instability in BRCA1 deficient cells.

Methods: We analysed DNA DSBs by ?H2AX, 53BP1, and pATM1981
foci and neutral comet assay, estrogen metabolite concentrations by LC-MS/MS,
and BRCA1 transcriptional regulation of metabolism genes by ChIP-chip, ChIP,
and qRT-PCR.

Results: We show that estrogen metabolism is perturbed in BRCA1 deficient
cells resulting in elevated production of 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), and decreased production of the protective metabolite
4-methoxyestradiol. We demonstrate that 2-OHE2 and 4-OHE2 treatment leads
to DNA double strand breaks (DSBs) in breast cells, and these DSBs were exacerbated
in both BRCA1 depleted cells and BRCA1 heterozygous cells (harbouring
185delAG mutation). Furthermore, the DSBs were not repaired efficiently in either
BRCA1 depleted or heterozygous cells, and we found that 2-OHE2 and 4-OHE2
treatment generates chromosomal aberrations in BRCA1 depleted cells. We suggest
that the increase in DNA DSBs in BRCA1 deficient cells is due to loss of
both BRCA1 transcriptional repression of estrogen metabolising genes (such as
CYP1A1 and CYP3A4) and loss of transcriptional activation of detoxification
genes (such as COMT).

Conclusions: We suggest that BRCA1 loss results in estrogen driven tumourigenesis
through a combination of increased expression of estrogen metabolising
enzymes and reduced expression of protective enzymes, coupled with a defect in
the repair of DNA DSBs induced by endogenous estrogen metabolites. The overall
effect being an exacerbation of genomic instability in estrogen regulated tissues in
BRCA1 mutation carriers.

Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of "simple" cancer-associated chromosome deletions.

Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally

Cytosolic phospholipase A2 (cPLA2) is thought to be the rate-limiting enzyme in the arachidonic acid/eicosanoid cascade. The ability of various agonists to increase steady-state cPLA2 mRNA levels has previously been reported. The current study delineates the contributions of transcriptional and post-transcriptional processes to the regulation of cPLA2 gene expression in response to a variety of agonists in cultured rat glomerular mesangial cells. Epidermal growth factor, platelet-derived growth factor, serum and phorbol myristate acetate all increase the half-life of cPLA2 mRNA transcripts, indicating a role for post-transcriptional modulation of gene expression. The presence of three ATTTA motifs in the 3' untranslated region (3'UTR) of the rat cPLA2 cDNA is ascertained. Heterologous expression of chimeric constructs with different 3'UTRs ligated into the 3' end of the luciferase coding region reveals that the presence of the cPLA2 3'UTR results in reduced luciferase activity compared with constructs without the cPLA2 3'UTR. Furthermore, the luciferase activity in the constructs with the cPLA2 3'UTR is increased in response to the same agonists which stabilize endogenous cPLA2 mRNA. A negligible effect of these agonists on transcriptional control of cPLA2 is evident using promoter-reporter constructs expressed in transient and stable transfectants. Taken together, these results indicate predominant post-transcriptional regulation of cPLA2 mRNA levels.

Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

New genetic loci link adipose and insulin biology to body fat distribution

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.

BRCA1, a 'complex' protein involved in the maintenance of genomic stability

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.

Uncovering BRCA1-regulated signalling pathways by microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Brca1 and brca2:Role in the DNA damage response, cancer formation and treatment

BRCA1 and BRCA2 are highly penetrant breast and ovarian cancer susceptibility genes that are mutated in a significant proportion of familial breast and ovarian cancer syndromes. Both of these genes are tumour suppressors, the products of which play vital roles in the cellular response to DNA damage. These proteins function in a number of cellular pathways in order to maintain genomic stability including DNA damage signaling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodeling, transcriptional regulation and apoptosis. This chapter will discuss the functions of these proteins and how they relate to tumour development, and therapy. © 2009 Springer Science+Business Media B.V.