The BRCA1 and BARD1 Association with the RNA Polymerase II Holoenzyme

Cancer Research. Aug, 2002  |  Pubmed ID: 12154023

We have previously shown that endogenous BRCA1 and overexpressed epitope-tagged BRCA1 are present in the transcription complex called the RNA polymerase II holoenzyme (holo-pol). In this study, we further characterized BRCA1 association with the holo-pol by overexpressing deletion mutants of epitope-tagged BRCA1. We found that BRCA1-associated RING domain protein (BARD1) is a component of the holo-pol complex. Deletion of the BRCA1 NH(2) terminus, which is bound by BARD1 as well as other proteins, eliminates >98% of BRCA1 association with the holo-pol. In contrast with earlier observations, deletion of the COOH terminus of BRCA1 did not affect significantly the association with holo-pol. Immunocytochemistry of expressed full-length and deletion mutants of BRCA1 showed that the NH(2) terminus of BRCA1 is important for nuclear dot formation in S-phase. An intact BRCA1 NH(2) terminus is required for the association with holo-pol and for subnuclear localization in S-phase foci. Taken together, these data support a role for BRCA1 regulation of holo-pol function.

Creating a Tool-kit for Exploring BRCA1 Function

Cancer Biology & Therapy. Sep-Oct, 2002  |  Pubmed ID: 12496478

Overexpression of a Protein Fragment of RNA Helicase A Causes Inhibition of Endogenous BRCA1 Function and Defects in Ploidy and Cytokinesis in Mammary Epithelial Cells

Oncogene. Feb, 2003  |  Pubmed ID: 12592385

The breast- and ovarian-specific tumor suppressor, BRCA1, has been implicated to function in many nuclear processes, including DNA damage repair, recombination, transcription, ubiquitination, cell cycle checkpoint enforcement, and centrosome regulation. Utilizing a previously described interaction between BRCA1 and RNA helicase A (RHA), we have developed a dominant-negative approach to block BRCA1 function in human breast epithelial cells. Overexpression of a truncated RHA peptide that can bind to the BRCA1 carboxy-terminus prevents normal BRCA1 function, such as BRCA1 association with nuclear foci following DNA damage. Overexpression of this dominant-negative protein induces pleomorphic nuclei, aberrant mitoses with extra centrosomes, and tetraploidy. This model system allows us to observe changes to mammary epithelial cells that occur acutely following loss of BRCA1 function. Furthermore, inhibition of BRCA1 via overexpressing the RHA fragment coincides with a reduction in PARP-1 protein expression, suggesting a possible mechanism for BRCA1 in the maintenance of genomic integrity.

The Multiple Nuclear Functions of BRCA1: Transcription, Ubiquitination and DNA Repair

Current Opinion in Cell Biology. Jun, 2003  |  Pubmed ID: 12787778

Interest in BRCA1 stems from its role as a tumour suppressor in breast and ovarian cancer. Intensive research in BRCA1 has revealed little about its specific role in cancer; rather, this protein has been implicated in a multitude of important cellular processes. The diverse biochemical activities of BRCA1 combine to protect the genome from damage. New data reveal that BRCA1 transcriptionally regulates some DNA-repair genes, and, in addition, new roles for BRCA1 have been identified in heterochromatin formation on the X chromosome, double-strand-break repair, and ubiquitination. These diverse activities of BRCA1 may be linked in a single pathway, or BRCA1 might function in multiple nuclear processes.

Transcription from the Perspective of the DNA: Twists and Bumps in the Road

Critical Reviews in Eukaryotic Gene Expression. 2003  |  Pubmed ID: 12839093

Although nature's design of the DNA double helix is ingenious for the storage of information, this design creates topological problems for the processes that occur on the DNA. Cellular processes, such as transcription, replication, chromosomal segregation, and chromosomal condensation, are all complicated by the double helix. The problem is compounded in cells since the DNA is in chromatin. Topoisomerases relax positive and negative superhelical turns in DNA, and thereby topoisomerases have long been recognized as key components of the DNA replication and chromosome segregation and condensation machinery. A role for topoisomerases in the transcription process has also been noted in living cells, but only recently has such a role been recapitulated in the test tube for transcription reactions. New data are discussed that demonstrate that for in vitro transcription reactions, topoisomerases are dispensable when the template is naked DNA, but when the template is reconstituted into chromatin, topoisomerases are required for transcription to proceed efficiently.

Elongation by RNA Polymerase II on Chromatin Templates Requires Topoisomerase Activity

Nucleic Acids Research. Sep, 2003  |  Pubmed ID: 12930951

Transcription on chromatin by RNA polymerase II (pol II) is repressed as compared with transcription on histone-free DNA. In this study, we show that human topoisomerase I (topo I) and yeast topoisomerase II (topo II), each of which relax both positive and negative superhelical tension, reverse the transcriptional repression by chromatin. In the presence of bacterial topo I, which can relax only negative superhelical tension, the transcription is repressed on chromatin templates. The data together show that the relaxation of positive superhelical tension by these enzymes was the key property required for RNA synthesis from chromatin templates. In the absence of topoisomerase, transcriptional repression on chromatin depended on RNA length. The synthesis of transcripts of 100 nt or shorter was unaffected by chromatin, but repression was apparent when the RNA transcript was 200 nt or longer. These findings suggest that transcription on chromatin templates results in the accumulation of positive superhelical tension by the elongating polymerase, which in turn inhibits further elongation in the absence of topoisomerase activity.

Phosphorylation of Histone H2A Inhibits Transcription on Chromatin Templates

The Journal of Biological Chemistry. May, 2004  |  Pubmed ID: 15010469

The regulation of gene expression via the histone code has, for the most part, revealed that histone modifications cause the recruitment of adaptor proteins that indirectly regulate the synthesis of RNA. Using purified factors to assemble and modify the chromatin and to transcribe the DNA, we investigated whether modifications of histones may directly impact the RNA polymerase II transcription process. We screened proteins known to modify histones for effects on transcription, and we found that the mitogen- and stress-induced kinase, MSK1, inhibited RNA synthesis. Inhibition of transcription by MSK1 was most sensitive when the template was in chromatin, as naked DNA templates were resistant to the effects of MSK1. We found that MSK1 phosphorylated histone H2A on serine 1, and mutation of serine 1 to alanine blocked the inhibition of transcription by MSK1. Furthermore, we found that acetylation of histone H3 by the p300 and CREB-binding protein associated factor, PCAF, suppressed the kinase-dependent inhibition of transcription. These results suggest that acetylation of histones may stimulate transcription by suppressing an inhibitory phosphorylation by a kinase as MSK1.

Expression of an Amino-terminal BRCA1 Deletion Mutant Causes a Dominant Growth Inhibition in MCF10A Cells

Oncogene. Jul, 2004  |  Pubmed ID: 15122325

Expression of deletion mutants of the breast and ovarian cancer-specific tumor suppressor protein, BRCA1, in the mammary epithelial cell line MCF10A revealed a powerful growth suppressive effect by a mutant that has the amino-terminal 302 amino acids deleted (DeltaN-BRCA1). The growth suppression is associated with an increase in apoptosis and amplification in centrosome number. The growth inhibitory effect of DeltaN-BRCA1 was not observed in cervical epithelial HeLa cells, suggesting that the phenotypes of BRCA1 mutant proteins differ depending on the cell line being tested. An internal domain, including BRCA1 residues 303-1292, caused the suppression of MCF10A cell growth, and the amino terminus of BRCA1 autoinhibited the growth suppression. Single point mutations that disrupted the amino-terminal RING domain of BRCA1 caused significant suppression of growth in MCF10A cells. These results suggest that the proper function of the RING domain, likely to be ubiquitin ligase function, is important in regulating the growth of the mammary epithelial cell line and in autoregulating the powerful internal growth-inhibiting domain of the BRCA1 tumor suppressor.

A Unified Nomenclature for Protein Subunits of Mediator Complexes Linking Transcriptional Regulators to RNA Polymerase II

Molecular Cell. Jun, 2004  |  Pubmed ID: 15175151

Overview of History and Progress in BRCA1 Research: the First BRCA1 Decade

Cancer Biology & Therapy. Jun, 2004  |  Pubmed ID: 15197353

The BRCA1 protein presents a paradox to the scientists investigating its link to cancer. BRCA1 provides a function essential to all cell types, but its mutation is only associated with a subset of cancers including breast and ovarian cancers. The priorities of BRCA1 research are the identification of the function of BRCA1, which is required in all cells, and of the function specific to breast and ovarian cells that causes their cancerous transformation. BRCA1 has been shown to be required for multiple processes including transcription, chromatin remodeling, DNA damage repair, and the regulation of centrosome duplication. How BRCA1 regulates these processes is unclear: is the BRCA1 protein a direct participant in the biochemistry of each process, or is the BRCA1 protein regulating the gene expression of proteins that directly regulate all of these processes? The ubiquity of BRCA1 in multiple cellular processes is difficult to square with the specificity of tumors with which it is associated.

BRCA1-dependent Ubiquitination of Gamma-tubulin Regulates Centrosome Number

Molecular and Cellular Biology. Oct, 2004  |  Pubmed ID: 15367667

Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including gamma-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of gamma-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate gamma-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of gamma-tubulin in vitro, and expression in cells of gamma-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.

The Tumor Suppressor Protein P53 Functions Similarly to P63 and P73 in Activating Transcription in Vitro

Cancer Biology & Therapy. Apr, 2005  |  Pubmed ID: 15846087

The p53 tumor suppressor protein functions via specific gene activation to inhibit passage through the cell cycle and to trigger apoptosis. The p53 protein is homologous to p63 and p73, proteins that regulate transcription via the same promoter sequences but which activate different genes. In this study we tested whether p53, p63, and p73 have different mechanisms of activating transcription and if such a difference could explain how each factor stimulates the transcription of distinct sets of genes. We found that when comparing p53 to the transcriptional activator, GAL4-VP16, both of which are classified as acidic activators, that stimulation of transcription by p53 is dependent upon low Mg2+ concentrations and limiting amounts of extract. By comparison, the stimulation of RNA synthesis by GAL4-VP16 was not dependent on a specific concentration of Mg2+ but did require higher amounts of extract, suggesting that a certain factor not required for p53-dependent gene activation was limiting in the extract. In contrast to the differences between p53 and GAL4-VP16, p63 and p73 both regulated transcription in vitro under similar conditions as did p53. All three proteins, purified to near homogeneity, were equally active in binding to the p53-response element, and equally active in stimulating transcription reactions using naked DNA templates, DNA templates reconstituted in chromatin using histones purified from HeLa cells, or hyper-acetylated histones. These results argue that the gene specificity of p63 and p73 dependent activation of transcription depends upon specific coactivators present in the specific cell types and upon other factors bound to the promoters.

Direct DNA Binding Activity of the Fanconi Anemia D2 Protein

The Journal of Biological Chemistry. Jun, 2005  |  Pubmed ID: 15849361

It is known that the Fanconi anemia D2 protein is vital for protecting the genome from DNA damage, but what activities this protein has are unknown. In these experiments we purified full-length Fanconi anemia protein D2 (FANCD2), and we found that FANCD2 bound to DNA with specificity for certain structures: double strand DNA ends and Holliday junctions. Proteins containing patient-derived mutations or artificial variants of the FANCD2 protein were similarly expressed and purified, and each variant bound to the Holliday junction DNA with similar affinity as did the wild-type protein. There was no single discrete domain of FANCD2 protein that bound to DNA, but rather the full-length protein was required for structure-specific DNA binding. This finding of DNA binding is the first biochemical activity identified for this key protein in the Fanconi anemia pathway.

Degradation of Cdt1 During S Phase is Skp2-independent and is Required for Efficient Progression of Mammalian Cells Through S Phase

The Journal of Biological Chemistry. Jun, 2005  |  Pubmed ID: 15855168

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854-30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807-30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.

BRCA1/BARD1 Ubiquitinate Phosphorylated RNA Polymerase II

The Journal of Biological Chemistry. Jul, 2005  |  Pubmed ID: 15886201

The breast- and ovarian-specific tumor suppressor BRCA1, when associated with BARD1, is an ubiquitin ligase. We have shown here that this heterodimer ubiquitinates a hyperphosphorylated form of Rpb1, the largest subunit of RNA polymerase II. Two major phosphorylation sites have been identified in the Rpb1 carboxyl terminal domain, serine 2 (Ser-2) or serine 5 (Ser-5) of the YSPTSPS heptapeptide repeat. Only the Ser-5 hyperphosphorylated form is ubiquitinated by BRCA1/BARD1. Overexpression of BRCA1 in cells stimulated the DNA damage-induced ubiquitination of Rpb1. Similar to the in vitro reaction, the stimulation of Rpb1 ubiquitination by BRCA1 in cells occurred only on those molecules hyperphosphorylated on Ser-5 of the heptapeptide repeat. In vitro, the carboxyl terminus of BRCA1 (amino acids 501-1863) was dispensable for the ubiquitination of hyperphosphorylated Rpb1. In cells, however, efficient Rpb1 ubiquitination required the carboxyl terminus of BRCA1, suggesting that interactions mediated by this region were essential in the complex milieu of the nucleus. These results link the BRCA1-dependent ubiquitination of the polymerase with DNA damage.

Centrosomal Microtubule Nucleation Activity is Inhibited by BRCA1-dependent Ubiquitination

Molecular and Cellular Biology. Oct, 2005  |  Pubmed ID: 16166645

In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of gamma-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of gamma-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of gamma-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.

Recruitment of ORC or CDC6 to DNA is Sufficient to Create an Artificial Origin of Replication in Mammalian Cells

Genes & Development. Dec, 2005  |  Pubmed ID: 16322558

Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.

Direct Stimulation of Transcription Initiation by BRCA1 Requires Both Its Amino and Carboxyl Termini

The Journal of Biological Chemistry. Mar, 2006  |  Pubmed ID: 16473884

Published experiments suggest that BRCA1 interaction with RNAPII and regulation of a number of target genes may be central to its role as a tumor suppressor. Previous in vivo and in vitro work has implicated the carboxyl terminus of BRCA1 in transcriptional stimulation, but the mechanism of action remains unknown, and whether the full-length protein stimulates transcription is controversial. BRCA1 interacts with a number of enhancer-binding transcriptional activators, suggesting that these factors recruit BRCA1 to promoters, where it stimulates RNA synthesis. To investigate whether BRCA1 has intrinsic transcriptional activity, we established a fully purified transcription assay. We demonstrate here that BRCA1 stimulates transcription initiation across a range of promoters. Both the amino and carboxyl termini of BRCA1 are required for this activity, but the BRCA1-binding partner, BARD1, is not. Our data support a model whereby BRCA1 stabilizes productive preinitiation complexes and thus stimulates transcription.

Substrates of the BRCA1-dependent Ubiquitin Ligase

Cancer Biology & Therapy. Feb, 2006  |  Pubmed ID: 16479151

Discovering the precise function of the breast and ovarian specific tumor suppressor, BRCA1, has proven to be quite complicated. It has been determined that BRCA1, together with BARD1, comprise an E3 ubiquitin ligase. Since it is now known that BRCA1 is an enzyme, the challenge for BRCA1 research is to learn how this enzymatic activity functions in normal breast and ovarian cells in order to suppress cancerous transformation. This review will survey the known ubiquitination substrates of BRCA1 and suggest how these reactions may influence the genomic stability and proliferation of breast cells.

BRCA1 DNA-binding Activity is Stimulated by BARD1

Cancer Research. Feb, 2006  |  Pubmed ID: 16489000

The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of BRCA1 in DNA repair.

Identification of Domains of BRCA1 Critical for the Ubiquitin-dependent Inhibition of Centrosome Function

Cancer Research. Apr, 2006  |  Pubmed ID: 16618730

The breast and ovarian cancer specific tumor suppressor BRCA1, bound to BARD1, has multiple functions aimed at maintaining genomic stability in the cell. We have shown earlier that the BRCA1/BARD1 E3 ubiquitin ligase activity regulates centrosome-dependent microtubule nucleation. In this study, we tested which domains of BRCA1 and BARD1 were required to control the centrosome function. In the present study, (a) we confirmed that the ubiquitination activity of BRCA1 regulates centrosome number and function in Hs578T breast cancer cells; (b) we observed that both the amino and carboxyl termini of BRCA1 are required for regulation of centrosome function in vitro; (c) an internal domain (770-1,290) is dispensable for centrosome regulation; (d) BARD1 is required for regulation of centrosome function and protein sequences within the terminal 485 amino acids are necessary for activity; and (e) BARD1 is localized at the centrosome throughout the cell cycle. We conclude that the BRCA1-dependent E3 ubiquitin ligase functions to restrain centrosomes in mammary cells, and loss of BRCA1 in the precancerous breast cell leads to centrosomal hypertrophy, a phenotype commonly observed in incipient breast cancer.

Oncoprotein EWS-FLI1 Activity is Enhanced by RNA Helicase A

Cancer Research. Jun, 2006  |  Pubmed ID: 16740692

RNA helicase A (RHA), a member of the DEXH box helicase family of proteins, is an integral component of protein complexes that regulate transcription and splicing. The EWS-FLI1 oncoprotein is expressed as a result of the chromosomal translocation t(11;22) that occurs in patients with the Ewing's sarcoma family of tumors (ESFT). Using phage display library screening, we identified an EWS-FLI1 binding peptide containing homology to RHA. ESFT cell lines and patient tumors highly expressed RHA. GST pull-down and ELISA assays showed that EWS-FLI1 specifically bound RHA fragment amino acids 630 to 1020, which contains the peptide region discovered by phage display. Endogenous RHA was identified in a protein complex with EWS-FLI1 in ESFT cell lines. Chromatin immunoprecipitation experiments showed both EWS-FLI1 and RHA bound to EWS-FLI1 target gene promoters. RHA stimulated the transcriptional activity of EWS-FLI1 regulated promoters, including Id2, in ESFT cells. In addition, RHA expression in mouse embryonic fibroblast cells stably transfected with EWS-FLI1 enhanced the anchorage-independent phenotype above that with EWS-FLI1 alone. These results suggest that RHA interacts with EWS-FLI1 as a transcriptional cofactor to enhance its function.

Centrosome Function in Normal and Tumor Cells

Journal of Cellular Biochemistry. Dec, 2006  |  Pubmed ID: 16817224

Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process.

Alpha-synuclein Acts in the Nucleus to Inhibit Histone Acetylation and Promote Neurotoxicity

Human Molecular Genetics. Oct, 2006  |  Pubmed ID: 16959795

Alpha-synuclein is a neuronal protein implicated genetically in Parkinson's disease. alpha-synuclein localizes to the nucleus and presynaptic nerve terminals. Here we show that alpha-synuclein mediates neurotoxicity in the nucleus. Targeting of alpha-synuclein to the nucleus promotes toxicity, whereas cytoplasmic sequestration is protective in both cell culture and transgenic Drosophila. Toxicity of alpha-synuclein can be rescued by administration of histone deacetylase inhibitors in both cell culture and transgenic flies. Alpha-synuclein binds directly to histones, reduces the level of acetylated histone H3 in cultured cells and inhibits acetylation in histone acetyltransferase assays. Alpha-synuclein mutations that cause familial Parkinson's disease, A30P and A53T, exhibit increased nuclear targeting in cell culture. These findings implicate nuclear alpha-synuclein in promoting nigrostriatal degeneration in Parkinson's disease and encourage exploration of histone deacetylase inhibitors as potential therapies for the disorder.

The BRCA1 E3 Ubiquitin Ligase Controls Centrosome Dynamics

Cell Cycle (Georgetown, Tex.). Sep, 2006  |  Pubmed ID: 16969086

The breast cancer specific tumor suppressor protein 1, BRCA1, mediates functions for all cells to grow. The puzzle of BRCA1 is that its loss is only associated with tumors in breast and ovarian epithelial cells. In this focused review, we highlight the data linking BRCA1 to the centrosome function, and we suggest that the specificity for breast tumors is due to a loss in restraint on centrosome function. Amplification of centrosome numbers secondary to loss of BRCA1 can drive the cell into the aneuploid state, thus, by this perspective loss of BRCA1 is a mutator phenotype.

A Mechanism for Transcriptional Repression Dependent on the BRCA1 E3 Ubiquitin Ligase

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2007  |  Pubmed ID: 17420471

Loss of function of the tumor suppressor protein BRCA1 is responsible for a high percentage of familial and also sporadic breast cancers. Early work identified a stimulatory transcriptional coactivator function for the BRCA1 protein, and more recently, BRCA1 has been implicated in transcriptional repression, although few examples of repressed genes have been characterized. We recently used an in vitro transcription assay to identify a biochemical mechanism that explained the BRCA1 stimulatory activity. In this study, we identified an ubiquitin-dependent mechanism by which BRCA1 inhibits transcription. BRCA1 ubiquitinates the transcriptional preinitiation complex, preventing stable association of TFIIE and TFIIH, and thus blocks the initiation of mRNA synthesis. What is striking about this mechanism of regulation by BRCA1 is that the ubiquitination of the preinitiation complex is not targeting proteins for degradation by the proteasome, nor are ubiquitin receptors modifying the activity, but rather the ubiquitin moiety itself interferes with the assembly of basal transcription factors at the promoter. Using RNAi to knockdown expression of the endogenous BRCA1 protein, we assessed the level of repression dependent on BRCA1 in the cell, and we found that BRCA1 is at least as significant a transcriptional repressor as it is an activator. These results define a biochemical mechanism by which the BRCA1 enzymatic activity regulates a key cellular process.

Aurora-A Kinase Regulates Breast Cancer Associated Gene 1 Inhibition of Centrosome-dependent Microtubule Nucleation

Cancer Research. Dec, 2007  |  Pubmed ID: 18056443

Breast cancer-associated gene 1 (BRCA1) regulates the duplication and the function of centrosomes in breast cells. We have previously shown that BRCA1 ubiquitin ligase activity directly inhibits centrosome-dependent microtubule nucleation. However, there is a paradox because centrosome microtubule nucleation potential is highest during mitosis, a phase when BRCA1 is most abundant at the centrosome. In this study, we resolve this conundrum by testing whether centrosomes from cells in M phase are regulated differently by BRCA1 when compared with other phases of the cell cycle. We observed that BRCA1-dependent inhibition of centrosome microtubule nucleation was high in S phase but was significantly lower during M phase. The cell cycle-specific effects of BRCA1 on centrosome-dependent microtubule nucleation were detected in living cells and in cell-free experiments using centrosomes purified from cells at specific stages of the cell cycle. We show that Aurora-A kinase modulates the BRCA1 inhibition of centrosome function by decreasing the E3 ubiquitin ligase activity of BRCA1. In addition, dephosphorylation of BRCA1 by protein phosphatase 1 alpha enhances the E3 ubiquitin ligase activity of BRCA1. These observations reveal that the inhibition of centrosome microtubule nucleation potential by the BRCA1 E3 ubiquitin ligase is controlled by Aurora-A kinase and protein phosphatase 1 alpha-mediated phosphoregulation through the different phases of the cell cycle.

BRCA1 Control of Steroid Receptor Ubiquitination

Science's STKE : Signal Transduction Knowledge Environment. Jun, 2007  |  Pubmed ID: 17579243

The ubiquitin ligase activity of the breast and ovarian cancer-associated tumor suppressor protein BRCA1, as part of a heterodimer with BARD1 (BRCA1-associated RING domain 1), is critical for its role as a tumor suppressor, but its targets, and the consequences of ubiquitin modification, by the BRCA1 protein complex are only now being discovered. BRCA1 is now known to control the ubiquitination of the estrogen and progesterone receptors, and although the consequences of the ubiquitination of these steroid receptors are unknown, the identification of these specific substrates suggests a link between the ubiquitin ligase activity of BRCA1 and its specificity as a tumor suppressor in breast and ovarian tissues.

Network Modeling Links Breast Cancer Susceptibility and Centrosome Dysfunction

Nature Genetics. Nov, 2007  |  Pubmed ID: 17922014

Many cancer-associated genes remain to be identified to clarify the underlying molecular mechanisms of cancer susceptibility and progression. Better understanding is also required of how mutations in cancer genes affect their products in the context of complex cellular networks. Here we have used a network modeling strategy to identify genes potentially associated with higher risk of breast cancer. Starting with four known genes encoding tumor suppressors of breast cancer, we combined gene expression profiling with functional genomic and proteomic (or 'omic') data from various species to generate a network containing 118 genes linked by 866 potential functional associations. This network shows higher connectivity than expected by chance, suggesting that its components function in biologically related pathways. One of the components of the network is HMMR, encoding a centrosome subunit, for which we demonstrate previously unknown functional associations with the breast cancer-associated gene BRCA1. Two case-control studies of incident breast cancer indicate that the HMMR locus is associated with higher risk of breast cancer in humans. Our network modeling strategy should be useful for the discovery of additional cancer-associated genes.

BRCA1 Regulates Gamma-tubulin Binding to Centrosomes

Cancer Biology & Therapy. Dec, 2007  |  Pubmed ID: 18087219

Centrosomes are the cellular organelles that nucleate microtubules (MTs) via the activity of gamma-tubulin ring complex(s) (gammaTuRC) bound to the pericentriolar material of the centrosomes. BRCA1, the breast and ovarian cancer specific tumor suppressor, inhibits centrosomal MT nucleation via its ubiquitin ligase activity, and one of the known BRCA1 substrates is the key gammaTuRC component, gamma-tubulin. We analyzed the mechanism by which BRCA1 regulates centrosome function using an in vitro reconstitution assay, which includes separately staged steps. Our results are most consistent with a model by which the BRCA1 ubiquitin ligase modifies both gamma-tubulin plus a second centrosomal protein that controls localization of gammaTuRC to the centrosome. We suggest that this second protein is an adapter protein or protein complex that docks gamma-TuRC to the centrosome. By controlling gamma-TuRC localization, BRCA1 appropriately inhibits centrosome function, and loss of BRCA1 would result in centrosome hyperactivity, supernumerary centrosomes and, possibly, aneuploidy.

Multiple Mechanisms Contribute to Inhibit Transcription in Response to DNA Damage

The Journal of Biological Chemistry. Apr, 2008  |  Pubmed ID: 18281289

Cellular DNA damage elicits the phosphorylation and ubiquitination of RNA polymerase II (RNAPII), leading to the global repression of transcription. In this report we show that there are at least two different pathways to transcriptional repression, depending on the type of DNA damage. After H2O2 treatment, transcription was rapidly inhibited and rapidly restored. On the other hand, UV irradiation caused a much slower transcriptional inhibition, with a corresponding depletion of unphosphorylated RNAPII. We found that after UV treatment, but not treatment with H2O2, the inhibition of transcription was dependent on both the proteasome and new protein synthesis. In addition, RNAPII activity and ubiquitination were regulated through the phosphorylation of RNAPII by the P-TEFb kinase. These results highlight that multiple cellular pathways exist to globally repress transcriptional processes that might interfere with the repair of DNA damage.

Regulation of Centrosomes by the BRCA1-dependent Ubiquitin Ligase

Cancer Biology & Therapy. Oct, 2008  |  Pubmed ID: 18927495

Centrosomes are the organelles that organize microtubule networks and establish the bipolar mitotic spindle, which is essential for the segregation of chromosomes during cell division. Proper duplication of centrosomes is necessary to prevent genetic instability, thus control of this organelle is important in the suppression of tumorigenesis. The BRCA1 dependent ubiquitination activity regulates centrosome number in breast derived cell lines and this activity is likely critical for the tumor suppression activity of BRCA1. This review will focus on the importance of controlling centrosome number and on the effect of BRCA1 on the centrosome duplication cycle in mammary cells.

The BRCA1-dependent Ubiquitin Ligase, Gamma-tubulin, and Centrosomes

Environmental and Molecular Mutagenesis. Oct, 2009  |  Pubmed ID: 19274767

Mutation of the breast and ovarian cancer specific tumor suppressor, BRCA1, results in supernumerary and hyperactive centrosomes, which in turn likely contribute to the aneuploidy evident in breast cancer cells. The BRCA1-dependent ubiquitin ligase activity is required for the regulation of centrosome function, and among its substrates is gamma-tubulin. Data suggest that during S and G2 phases of the cell cycle, the normal function of BRCA1 directs the ubiquitination of gamma-tubulin, resulting in inhibition of centrosome microtubule nucleation function and blocking of centrosome reduplication. Loss of BRCA1 activity, as occurs in breast cancer cells, would result in centrosomes that are unrestrained, leading to the hyperactive and over-duplicated centrosomes often observed in breast cancer cells. The current knowledge of BRCA1 regulation of centrosomes will be discussed in this focused review, and it will be suggested that this function is important for the tumor suppression phenotype of BRCA1.

Comparative Study on ChIP-seq Data: Normalization and Binding Pattern Characterization

Bioinformatics (Oxford, England). Sep, 2009  |  Pubmed ID: 19561022

Antibody-based Chromatin Immunoprecipitation assay followed by high-throughput sequencing technology (ChIP-seq) is a relatively new method to study the binding patterns of specific protein molecules over the entire genome. ChIP-seq technology allows scientist to get more comprehensive results in shorter time. Here, we present a non-linear normalization algorithm and a mixture modeling method for comparing ChIP-seq data from multiple samples and characterizing genes based on their RNA polymerase II (Pol II) binding patterns.

Cdk1 Participates in BRCA1-dependent S Phase Checkpoint Control in Response to DNA Damage

Molecular Cell. Aug, 2009  |  Pubmed ID: 19683496

Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging treatments by disrupting BRCA1 function.

Enabling Data Analysis on High-Throughput Data in Large Data Depository Using Web-Based Analysis Platform - A Case Study on Integrating QUEST with GenePattern in Epigenetics Research

Proceedings. IEEE International Conference on Bioinformatics and Biomedicine. Nov, 2009  |  Pubmed ID: 21151835

Enabling data analysis in large data depositories for high throughput experimental data such as gene microarrays and ChIP-seq is challenging. In this paper, we discuss three methods for integrating QUEST, a data depository for epigenetic experiments, with a web-based data analysis platform GenePattern. These methods are universal and can serve as an exemplary implementation resolving the dilemma facing many similar database systems in integrating data analysis tools.

BRCA1/BARD1 E3 Ubiquitin Ligase Can Modify Histones H2A and H2B in the Nucleosome Particle

Journal of Biomolecular Structure & Dynamics. Feb, 2010  |  Pubmed ID: 19916563

BRCA1, the protein product of the Breast Cancer Susceptibility Gene (BRCA1) has been implicated in multiple pathways that preserve genome stability, including cell cycle control, DNA repair, transcription, and chromatin remodeling. BRCA1, in complex with another RING-domain protein BARD1, possesses ubiquitin-ligase activity. Only a few targets for this activity have been identified in vivo. Nucleosomal histones may also be targets in vivo since they can be modified by the BRCA1/BARD1 complex in vitro. Here we demonstrate that the BRCA1/BARD1 complex can ubiquitylate both free H2A and H2B histones and histones in the context of nucleosomal particles. These results raise the possibility that BRCA1/BARD1 can directly affect nucleosomal structure, dynamics, and function through its ability to modify nucleosomal histones.

Identification of Breast Tumor Mutations in BRCA1 That Abolish Its Function in Homologous DNA Recombination

Cancer Research. Feb, 2010  |  Pubmed ID: 20103620

Effects of breast cancer-associated gene 1 (BRCA1) missense mutations on the function of BRCA1 protein in DNA recombination have been little studied. In this report, we adapted a homology-directed recombination (HDR) assay to analyze the effects of BRCA1 mutations on this function. Using a HeLa-derived cell line with a genomically integrated recombination substrate, we expressed an endonuclease creating a double-stranded break in the substrate that the HDR assay scores by generation of green fluorescent protein-positive cells. By combining RNA interference (RNAi) that targets cellular BRCA1 mRNA with expression of RNAi-resistant BRCA1 mutants, we could effectively substitute selected point mutants to test these in the cellular recombination assay. We found that approximately 300 residues at both termini of the BRCA1 protein were essential for HDR. Whereas some mutations analyzed were neutral, mutations that altered any zinc-coordinating residue or generated M18T and T37R alterations were defective for recombination. This study established a robust assay system to analyze the function of BRCA1 in regulating homologous recombination, which is critical for its tumor suppressor function.

BRCA1 Represses Amphiregulin Gene Expression

Cancer Research. Feb, 2010  |  Pubmed ID: 20103632

BRCA1, the breast cancer- and ovarian cancer-specific tumor suppressor, can be a transcriptional repressor or a transcriptional activator, depending on the promoter context. To identify the genes activated or repressed by BRCA1, we have analyzed microarray results from cells depleted of BRCA1 and revealed a number of genes regulated by BRCA1 on the level of transcription. Among the genes repressed by BRCA1, we have identified amphiregulin (AREG) and early growth response-1 (EGR1). Results indicate that BRCA1 regulates AREG transcription directly through binding to the AREG promoter, however, we could not detect BRCA1 on the EGR1 promoter, suggesting that EGR1 is indirectly regulated by BRCA1. In an attempt to identify the mechanism of the AREG transcriptional repression by BRCA1, we have mapped two independent BRCA1 response elements on the AREG located at positions -202/-182 and +19/+122. BRCA1 depletion leads to induction of the AREG protein. Taken together, our data build the connection between BRCA1 loss of function and AREG upregulation-a change in gene expression often observed in breast cancer.

PI 3 Kinase Related Kinases-independent Proteolysis of BRCA1 Regulates Rad51 Recruitment During Genotoxic Stress in Human Cells

PloS One. 2010  |  Pubmed ID: 21103343

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.

Inference of Hierarchical Regulatory Network of Estrogen-dependent Breast Cancer Through ChIP-based Data

BMC Systems Biology. 2010  |  Pubmed ID: 21167036

Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF) partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks.

Histone Deacetylases 9 and 10 Are Required for Homologous Recombination

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21247901

We tested the role of histone deacetylases (HDACs) in the homologous recombination process. A tissue-culture based homology-directed repair assay was used in which repair of a double-stranded break by homologous recombination results in gene conversion of an inactive GFP allele to an active GFP gene. Our rationale was that hyperacetylation caused by HDAC inhibitor treatment would increase chromatin accessibility to repair factors, thereby increasing homologous recombination. Contrary to expectation, treatment of cells with the inhibitors significantly reduced homologous recombination activity. Using RNA interference to deplete each HDAC, we found that depletion of either HDAC9 or HDAC10 specifically inhibited homologous recombination. By assaying for sensitivity of cells to the interstrand cross-linker mitomycin C, we found that treatment of cells with HDAC inhibitors or depletion of HDAC9 or HDAC10 resulted in increased sensitivity to mitomycin C. Our data reveal an unanticipated function of HDAC9 and HDAC10 in the homologous recombination process.

Fanconi Anemia Protein FANCD2 Inhibits TRF1 PolyADP-ribosylation Through Tankyrase1-dependent Manner

Genome Integrity. 2011  |  Pubmed ID: 21314979

Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by developmental abnormalities, progressive bone marrow failure, and predisposition to cancer. The key FA protein FANCD2 crosstalks with members of DNA damage and repair pathways that also play a role at telomeres. Therefore, we investigated whether FANCD2 has a similar involvement at telomeres.

Comparing Multiple ChIP-sequencing Experiments

Journal of Bioinformatics and Computational Biology. Apr, 2011  |  Pubmed ID: 21523932

New high-throughput sequencing technologies can generate millions of short sequences in a single experiment. As the size of the data increases, comparison of multiple experiments on different cell lines under different experimental conditions becomes a big challenge. In this paper, we investigate ways to compare multiple ChIP-sequencing experiments. We specifically studied epigenetic regulation of breast cancer and the effect of estrogen using 50 ChIP-sequencing data from Illumina Genome Analyzer II. First, we evaluate the correlation among different experiments focusing on the total number of reads in transcribed and promoter regions of the genome. Then, we adopt the method that is used to identify the most stable genes in RT-PCR experiments to understand background signal across all of the experiments and to identify the most variable transcribed and promoter regions of the genome. We observed that the most variable genes for transcribed regions and promoter regions are very distinct. Gene ontology and function enrichment analysis on these most variable genes demonstrate the biological relevance of the results. In this study, we present a method that can effectively select differential regions of the genome based on protein-binding profiles over multiple experiments using real data points without any normalization among the samples.

KIAA0101 Interacts with BRCA1 and Regulates Centrosome Number

Molecular Cancer Research : MCR. Aug, 2011  |  Pubmed ID: 21673012

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.

BRCA1 Contributes to Transcription-coupled Repair of DNA Damage Through Polyubiquitination and Degradation of Cockayne Syndrome B Protein

Cancer Science. Oct, 2011  |  Pubmed ID: 21756275

BRCA1 is an important gene involved in susceptibility to breast and ovarian cancer and its product regulates the cellular response to DNA double-strand breaks. Here, we present evidence that BRCA1 also contributes to the transcription-coupled repair (TCR) of ultraviolet (UV) light-induced DNA damage. BRCA1 immediately accumulates at the sites of UV irradiation-mediated damage in cell nuclei in a manner that is fully dependent on both Cockayne syndrome B (CSB) protein and active transcription. Suppression of BRCA1 expression inhibits the TCR of UV lesions and increases the UV sensitivity of cells proficient in TCR. BRCA1 physically interacts with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiquitination and subsequent degradation of CSB occur following UV irradiation, even in the absence of Cockayne syndrome A (CSA) protein. The depletion of BRCA1 expression increases the UV sensitivity of CSA-deficient cells. These results indicate that BRCA1 is involved in TCR and that a BRCA1-dependent polyubiquitination pathway for CSB exists alongside the CSA-dependent pathway to yield more efficient excision repair of lesions on the transcribed DNA strand.