Identification and Characterization of a Transcriptional Silencer Upstream of the Human BRCA2 Gene

Sharan, Chakradhari; Hamilton, Nalo; Parl, Angelika K.; Singh, Pramod K.; Chaudhuri, Gautam

doi:10.1006/bbrc.1999.1652

Cited by 29 publications

(48 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The transcription factor binding sites from these regions and the associated transcription factors that contribute to regulation of the BRCA2 promoter are not yet known. Recently a repression domain associated with 10-fold downregulation of the BRCA2 promoter was reported (36). This domain is associated with two Alu repeats and is located in the Del-7 and Del-8 clones and is deleted from the Del-9 clone shown in Fig.…”

Section: Fig 3 Nuclear Factor B Induces the Brca2 Promotermentioning

confidence: 92%

Induction of the BRCA2 Promoter by Nuclear Factor-κB

Wu¹,

Jiang²,

Thangaraju³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

BRCA2 is a tumor suppressor gene that has been implicated in response to DNA damage, cell cycle control, and transcription. BRCA2 has been found to be overexpressed in many breast tumors, suggesting that altered expression of the BRCA2 gene may contribute to breast tumorigenesis. To determine how BRCA2 is overexpressed in tumors, we investigated the transcriptional regulation of the BRCA2 promoter. Deletion mapping of the BRCA2 promoter identified three regions associated with 3-fold activation or repression and one upstream stimulatory factor binding site associated with 20-fold activation. Gel shift and cotransfection studies verified the role of USF in regulation of BRCA2 transcription. Analysis of the ؊144 to ؊59 region associated with 3-fold activation identified a putative NFB binding site. Cotransfection of the p65 and p50 subunits of NFB upregulated the BRCA2 promoter 16-fold in a luciferase reporter assay, whereas mutations in the binding site ablated the effect. Gel shift and supershift assays with anti-p65 and -p50 antibodies demonstrated that NFB binds specifically to the NFB site. In addition, ectopic expression of NFB resulted in increased levels of endogeneous BRCA2 expression. Thus, NFB and USF regulate BRCA2 expression through the BRCA2 promoter.BRCA2 is a tumor suppressor gene associated with familial predisposition to breast and ovarian cancer (1, 2). Mutations in BRCA2 are thought to account for 20 -35% of all inherited breast cancers and are associated with a 37-85% lifetime risk of developing cancer (3, 4). The great majority of disease-associated mutations in BRCA2 result in truncation of the BRCA2 protein, suggesting that loss of function of BRCA2 results in tumor susceptibility. However, the mechanisms by which the BRCA2 protein suppresses tumor cell growth are largely unknown.The BRCA2 gene encodes a 3418-amino acid nuclear protein (2, 5), that has been implicated in the cellular response to DNA damage. BRCA2 interacts directly with RAD51, a protein involved in meiotic and mitotic recombination, DNA doublestranded break repair, and chromosome segregation (6, 7), through the BRC repeats and a C-terminal binding site. BRCA2Ϫ/Ϫ animals die as early embryos (8 -11), and viable BRCA2 Ϫ/Ϫ early mouse embryos are highly sensitive to ␥-irradiation-induced DNA damage (9). Moreover, cells expressing mutant BRCA2 are more sensitive to methyl methanesulfonate-induced DNA damage than cells expressing wild type BRCA2 (12), and BRCA2 appears to be required for ionizing radiation-induced assembly of a RAD51 protein complex in vivo (13).BRCA2 may be also involved in regulation of the cell cycle and genome instability. BRCA2 is expressed in a cell cycle-dependent manner with peak expression in the S and G 2 phases of the cell cycle. Low levels of expression are detected in G 0 , G 1 , and M phase (14). Cell cycle-dependent expression has recently been associated with binding of the upstream stimulatory factor (USF) 1 protein and Elf-1 transcription factor to the BRCA2 promoter (15). In addition, BRCA2 ex...

show abstract

Section: Fig 3 Nuclear Factor B Induces the Brca2 Promotermentioning

confidence: 92%

Induction of the BRCA2 Promoter by Nuclear Factor-κB

Wu¹,

Jiang²,

Thangaraju³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…HMEC cells were grown in medium purchased from Clonetics under their recommended conditions. Other cells were maintained and grown in ATCC recommended media and conditions, as described before (12,13). Resting or nondividing cells are defined as the cells that are in a 100% confluent state of growth in a culture flask or the cells that are arrested mostly at the G 0 phase by serum starvation.…”

Section: Methodsmentioning

confidence: 99%

“…Protein lysates were prepared from the cells, and luciferase activities were measured as described previously (12). Firefly luciferase activity was normalized with respect to Renilla luciferase activity and presented as a ratio (relative light units).…”

Section: Methodsmentioning

confidence: 99%

“…Electerophoretic Mobility Shift Assay-Double-stranded DNA containing the 221-bp silencer fragment was labeled with [␥-32 P]ATP and used in electrophoretic mobility shift assay (12). Double-stranded DNA probes were purified from the reaction mixture using a Bio-Gel P-100 column (Bio-Rad), incubated with whole cell extract from breast cells, and separated on 5% polyacrylamide gels as described previously (12,17).…”

Section: Methodsmentioning

confidence: 99%

“…We previously reported a 221-bp silencer sequence located at 700 bp upstream of the BRCA2 gene transcription start site (12) (see Fig. 1A).…”

mentioning

confidence: 90%

See 2 more Smart Citations

Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Tripathi

Misra

Khedkar

et al. 2005

Journal of Biological Chemistry

Self Cite

137

View full text Add to dashboard Cite

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the nondividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the ؊701 to ؊921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

show abstract

Implications of the Human Genome for Understanding Human Biology and Medicine

Subramanian¹

2001

JAMA

128

View full text Add to dashboard Cite

Clinical researchers, practicing physicians, patients, and the general public now live in a world in which the 2.9 billion nucleotide codes of the human genome are available as a resource for scientific discovery. Some of the findings from the sequencing of the human genome were expected, confirming knowledge presaged by many decades of research in both human and comparative genetics. Other findings are unexpected in their scientific and philosophical implications. In either case, the availability of the human genome is likely to have significant implications, first for clinical research and then for the practice of medicine. This article provides our reflections on what the new genomic knowledge might mean for the future of medicine and how the new knowledge relates to what we knew in the era before the availability of the genome sequence. In addition, practicing physicians in many communities are traditionally also ambassadors of science, called on to translate arcane data or the complex ramifications of biology into a language understood by the public at large. This article also may be useful for physicians who serve in this capacity in their communities. We address the following issues: the number of protein-coding genes in the human genome and certain classes of noncoding repeat elements in the genome; features of genome evolution, including large-scale duplications; an overview of the predicted protein set to highlight prominent differences between the human genome and other sequenced eukaryotic genomes; and DNA variation in the human genome. In addition, we show how this information lays the foundations for ongoing and future endeavors that will revolutionize biomedical research and our understanding of human health.

show abstract

Identification and Characterization of a Transcriptional Silencer Upstream of the Human BRCA2 Gene

Cited by 29 publications

References 16 publications

Induction of the BRCA2 Promoter by Nuclear Factor-κB

Induction of the BRCA2 Promoter by Nuclear Factor-κB

Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Implications of the Human Genome for Understanding Human Biology and Medicine

Contact Info

Product

Resources

About