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.

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.

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Here we show that complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor mle. In transient transfection assays, RHA was found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. As a mutation in RHA that compromised its helicase activity correspondingly reduced CREB-dependent transcription, we propose that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters.

The familial breast-ovarian tumor suppressor gene product BRCA1 was found to be a component of the RNA polymerase II holoenzyme by several criteria. BRCA1 was found to copurify with the holoenzyme over multiple chromatographic steps. Other tested transcription activators that could potentially contact the holoenzyme were not stably associated with the holoenzyme as determined by copurification. Antibody specific for the holoenzyme component hSRB7 specifically purifies BRCA1. Immunopurification of BRCA1 complexes also specifically purifies transcriptionally active RNA polymerase II and transcription factors TFIIF, TFIIE, and TFIIH. Moreover, a BRCA1 domain, which is deleted in about 90% of clinically relevant mutations, participates in binding to the holoenzyme complex in cells. These data are consistent with recent data identifying transcription activation domains in the BRCA1 protein and link the BRCA1 tumor suppressor protein with the transcription process as a holoenzyme-bound protein.BRCA1 is a tumor suppressor gene that is mutated in a significant fraction of cases of inherited breast and ovarian cancer. Approximately 3% of breast cancer is attributable to inherited mutations in BRCA1. Indeed, in ϳ50% of families with an abnormally high incidence of breast cancer through multiple generations, the offending mutation is in the BRCA1 gene (1-3). The BRCA1 product is likely to have tumor suppression function, since tumors arising in members of BRCA1-linked families show loss of heterozygosity at the BRCA1 locus, with retention of the mutant͞disease-predisposing allele (4, 5).The BRCA1 gene encodes a 1,863-amino acid protein without extensive homology to other proteins (1, 2). The primary sequence is noteworthy for a RING-finger motif and an acidic carboxyl terminus (1), both of which are characteristics of certain transcription factors. The BRCA1 7.8-kb mRNA is observed in many tissues, with expression highest in testis and thymus (1). About 90% of the mutations observed in the BRCA1 gene result in truncations, and the remainder of clinically relevant mutations are individual missense abnormalities that are scattered along the entire coding unit (3).A defined carboxyl-terminal segment of BRCA1 can activate transcription when fused to the DNA binding domain of GAL4 (6, 7). These fusion proteins activated transcription from promoters containing a GAL4 binding site. Importantly, fusion proteins bearing clinically relevant point mutations were inactive in this assay, implying, at a minimum, that the transcription assay is a faithful monitor of the native structure of a segment of the protein. Although there are other interpretations, these data have licensed the speculation that BRCA1 is, at least in part, a transcription factor. Whatever the significance of the transactivation potential of its carboxylterminal region, there is now evidence pointing to a role for BRCA1 in the control of DNA repair and genome stability (8). Hence, if it proves to have genuine transcription regulation function, it will be int...

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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 ␥-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of ␥-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate ␥-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of ␥-tubulin in vitro, and expression in cells of ␥-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.Cancer cells frequently have unstable numbers of chromosomes (reviewed in reference 20). One mechanism for chromosomal instability is improper centrosome duplication, since the centrosome is the organelle that organizes the spindle for separation of chromosomes during mitosis. The presence of more than two centrosomes in a cell can result in lost or fragmented chromosomes after cell division. Human tumors derived from breast and other tissues have abnormal centrosome numbers in early-stage lesions. As an example, abnormal centrosome numbers have been detected in ductal carcinoma in situ, the first stage of breast cancer (21, 33), and BRCA1 has been shown to have a role in regulating centrosome number (reviewed in reference 9).BRCA1 is a tumor suppressor that is mutated in inherited breast and ovarian cancer cases, and it is also epigenetically down-regulated in sporadic breast cancers. Strikingly, BRCA1 function is required for nearly all cell types to grow; it has many roles in the cell. These functions include the regulation of DNA damage repair, transcription, and X-chromosome inactivation (reviewed in references 37 and 41). All of these processes could be important in protecting mammary cells from uncontrolled growth, but it is not clear why loss of BRCA1 specifically results in breast and ovarian cancer.There is growing evidence that BRCA1 functions as a regulator of centrosome number. First, BRCA1 is localized to the centrosome in mitotic cells (17,23). Second, interference with BRCA1 function by various methods can cause an increased centrosome number. For example, mouse fibroblasts derived from BRCA1 exon 11 knockouts have ...

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.KEYWORDS deep mutational scanning; BRCA1; variants of uncertain significance; human genetic variation; protein function I N an era of increasingly widespread genetic testing, DNA sequencing identifies many missense substitutions with unknown effects on protein function and disease risk. In the absence of genetic evidence, experimental measurement is the most reliable way to determine the functional impact of a variant of uncertain significance (VUS). However, initiating an experiment for each new variant observed in a gene is often impractical. When experiments are done, they are nearly always performed in a retrospective manner (Bouwman et al. 2013), such that the resulting data are not useful for the patient in whom the VUS was observed.By prospectively measuring, in a high-throughput fashion, the consequences of all possible missense mutations on a gene's function, we can generate a look-up table for interpreting newly observed VUS. Although functional analysis at this scale is made possible by deep mutational scanning (Fowler and Fields 2014), a central challenge is that any single assay may not recapitulate all the activities of a given protein in human disease. To address this challenge, we hypothesized that integrating the results of assays of multiple biochemical functions would strengthen estimates of the effects of mutations on disease risk (strategy outlined in Figure 1A). As a proof-ofconcept, we initiated massively parallel functional analysis of BRCA1, a protein for which there are multiple biochemical functions as well as known pathogenic and benign missense substitutions to benchmark results.BRCA1 has been subject to intense study since its implication in hereditary, early onset breast and ovarian cancer (Miki et al. 1994). All missense substitutions in BRCA1 that are known to be pathogenic occur in either the amino-terminal RING domain or the carboxy-terminal BRCT repeat (http:// brca.iarc.fr/LOVD/home.php?select_db=BRCA1). Although the RING domain represents only 5% of the BRCA1 protein, 58% of the pathogenic missense substitutions occur within this domain. Sixty-two missense substitutions in the RING domain have been observed in patient...

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