Predictive Factors for BRCA1 and BRCA2 Genetic Testing in an Asian Clinic-Based Population.

PURPOSE: The National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population. METHODS: A total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis. RESULTS: Of 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age (40 vs. 48 years, P=0.008). Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients (38 vs. 46 years, P=0.028). Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations. CONCLUSIONS: Our study provides evidence that TNBC or ER-negative patients may benefit from BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients.

Chromatin-Associated Proteins Revealed by SILAC-Proteomic Analysis Exhibit a High Likelihood of Requirement for Growth Fitness under DNA Damage Stress.

Chromatin-associated nonhistone proteins (CHRAPs) are readily collected from the DNaseI digested crude chromatin preparation. In this study, we show that the absolute abundance-based label-free quantitative proteomic analysis fail to identify potential CHRAPs from the CHRAP-prep. This is because that the most-highly abundant cytoplasmic proteins such as ribosomal proteins are not effectively depleted in the CHRAP-prep. Ribosomal proteins remain the top-ranked abundant proteins in the CHRAP-prep. On the other hand, we show that relative abundance-based SILAC-mediated quantitative proteomic analysis is capable of discovering the potential CHRAPs in the CHRAP-prep when compared to the whole-cell-extract. Ribosomal proteins are depleted from the top SILAC ratio-ranked proteins. In contrast, nucleus-localized proteins or potential CHRAPs are enriched in the top SILAC-ranked proteins. Consistent with this, gene-ontology analysis indicates that CHRAP-associated functions such as transcription, regulation of chromatin structures, and DNA replication and repair are significantly overrepresented in the top SILAC-ranked proteins. Some of the novel CHRAPs are confirmed using the traditional method. Notably, phenotypic assessment reveals that the top SILAC-ranked proteins exhibit the high likelihood of requirement for growth fitness under DNA damage stress. Taken together, our results indicate that the SILAC-mediated proteomic approach is capable of determining CHRAPs without prior knowledge.

Genomic binding profiling of the fission yeast stress-activated MAPK Sty1 and the bZIP transcriptional activator Atf1 in response to H2O2.

BACKGROUND: The evolutionally conserved MAPK Sty1 and bZIP transcriptional activator Atf1 are known to play a pivotal role in response to the reactive oxygen species in S. pombe. However, it is unclear whether all of the H(2)O(2)-induced genes are directly regulated by the Sty1-Atf1 pathway and involved in growth fitness under H(2)O(2)-induced stress conditions. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the study on ChIP-chip mapping of the genomic binding sites for Sty1, Atf1, and the Atf1's binding partner Pcr1; the genome-wide transcriptional profiling of the atf1 and pcr1 strains in response to H(2)O(2); and the phenotypic assessment of approximately 90 Atf1/Pcr1-bound or unbound genes for growth fitness under H(2)O(2) conditions. ChIP-chip analysis shows that Atf1 and Pcr1 binding sites are overlapped in the genome and constitutively present before H(2)O(2) stress. On the other hand, Sty1 recruitment primarily occurs at the Atf1/Pcr1 binding sites and is induced by H(2)O(2). We found that Atf1/Pcr1 is clearly responsible for the high-level transcriptional response to H(2)O(2). Furthermore, phenotypic assessment indicates that among the H(2)O(2)-induced genes, Atf1/Pcr1-bound genes exhibit a higher likelihood of functional requirement for growth fitness under the stress condition than the Atf1/Pcr1-unbound genes do. Notably, we found that the Atf1/Pcr1-bound genes regardless of their responsiveness to H(2)O(2) show a high probability of requirement for growth fitness. CONCLUSION/SIGNIFICANCE: Together, our analyses on global mapping of protein binding sites, genome-wide transcriptional profiling, and phenotypic assessment provide insight into mechanisms for global transcriptional regulation by the Sty1-Atf1 pathway in response to H(2)O(2)-induced reactive oxygen species.

A Cds1-mediated checkpoint protects the MBF activator Rep2 from ubiquitination by anaphase-promoting complex/cyclosome-Ste9 at S-phase arrest in fission yeast.

Transcription of the MluI cell cycle box (MCB) motif-containing genes at G(1) phase is regulated by the MCB-binding factors (MBF) (also called DSC1) in Schizosaccharomyces pombe. Upon S-phase arrest, the MBF transcriptional activity is induced through the accumulation of the MBF activator Rep2. In this study, we show that the turnover of Rep2 is attributable to ubiquitin-mediated proteolysis. Levels of Rep2 oscillate during the cell cycle, with a peak at G(1) phase, coincident with the MBF activity. Furthermore, we show that Rep2 ubiquitination requires the function of the E3 ligase anaphase-promoting complex/cyclosome (APC/C). Ste9 can be phosphorylated by the checkpoint kinase Cds1 in vitro, and its inhibition/phosphorylation at S-phase arrest is dependent on the function of Cds1. Our data indicate that the Cds1-dependent stabilization of Rep2 is achieved through the inhibition/phosphorylation of APC/C-Ste9 at the onset of S-phase arrest. Stabilization of Rep2 is important for stimulating transcription of the MBF-dependent genes to ensure a sufficient supply of proteins essential for cell recovery from S-phase arrest. We propose that oscillation of Rep2 plays a role in regulation of periodic transcription of the MBF-dependent genes during cell cycle progression.

Differential regulation of FLOWERING LOCUS C expression by vernalization in cabbage and Arabidopsis.

Vernalization is required to induce flowering in cabbage (Brassica oleracea var Capitata L.). Since FLOWERING LOCUS C (FLC) was identified as a major repressor of flowering in the vernalization pathway in Arabidopsis (Arabidopsis thaliana), two homologs of AtFLC, BoFLC3-2 and BoFLC4-1, were isolated from cabbage to investigate the molecular mechanism of vernalization in cabbage flowering. In addition to the sequence homology, the genomic organization of cabbage FLC is similar to that of AtFLC, except that BoFLC has a relatively smaller intron 1 compared to that of AtFLC. A vernalization-mediated decrease in FLC transcript level was correlated with an increase in FT transcript level in the apex of cabbage. This observation is in agreement with the down-regulation of FT by FLC in Arabidopsis. Yet, unlike that in Arabidopsis, the accumulation of cabbage FLC transcript decreased after cold treatment of leafy plants but not imbibed seeds, which is consistent with the promotion of cabbage flowering by vernalizing adult plants rather than seeds. To further dissect the different regulation of FLC expression between seed-vernalization-responsive species (e.g. Arabidopsis) and plant-vernalization-responsive species (e.g. cabbage), the pBoFLC4-1BoFLC4-1GUS construct was introduced into Arabidopsis to examine its vernalization response. Down-regulation of the BoFLC4-1GUS construct by seed vernalization was unstable and incomplete; in addition, the expression of BoFLC4-1GUS was not suppressed by vernalization of transgenic rosette-stage Arabidopsis plants. We propose a hypothesis to illustrate the distinct mechanism by which vernalization regulates the expression of FLC in cabbage and Arabidopsis.