Smc5/6's multifaceted DNA binding capacities stabilize branched DNA structures.

Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, and junction DNA formed by juxtaposed ss- and dsDNA, using correlative single-molecule fluorescence and force microscopy. We find that Smc5/6 displays distinct behaviors toward different types of DNA, dynamically associating with dsDNA while stably binding to junction DNA. Mechanistically, both the Nse1-3-4 subcomplex and ATP binding enhance the complex's dsDNA association. In contrast, Smc5/6's assembly onto ssDNA emanating from junction DNA, which occurs even in the presence high-affinity ssDNA binders, is aided by Nse1-3-4, but not by ATP. Moreover, we show that Smc5/6 protects junction DNA stability by preventing ssDNA annealing. The multifaceted DNA association behaviors of Smc5/6 provide a framework for understanding its diverse functions in genome maintenance and viral DNA restriction.

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA.

Biomolecular condensation constitutes an emerging mechanism for transcriptional regulation. Recent studies suggest that the co-condensation between transcription factors (TFs) and DNA can generate mechanical forces driving genome rearrangements. However, the reported forces generated by protein-DNA co-condensation are typically below one piconewton (pN), questioning its physiological significance. Moreover, the force-generating capacity of these condensates in the chromatin context remains unknown. Here, we show that Sox2, a nucleosome-binding pioneer TF, forms co-condensates with DNA and generates forces up to 7 pN, exerting considerable mechanical tension on DNA strands. We find that the disordered domains of Sox2 are required for maximum force generation but not for condensate formation. Furthermore, we show that nucleosomes dramatically attenuate the mechanical stress exerted by Sox2 by sequestering it from coalescing on bare DNA. Our findings reveal that TF-mediated DNA condensation can exert significant mechanical stress on the genome which can nonetheless be attenuated by the chromatin architecture.

Discovering drugs to overcome chemoresistance in ovarian cancers based on the cancer genome atlas tumor transcriptome profile.

Ovarian cancer accounts for the highest mortality among gynecologic cancers, mainly due to intrinsic or acquired chemoresistance. While mechanistic-based methods have been used to identify compounds that can overcome chemoresistance, an effective comprehensive drug screening has yet to be developed. We applied a transcriptome based drug sensitivity prediction method, to the Cancer Genome Atlas (TCGA) ovarian cancer dataset to impute patient tumor response to over 100 different drugs. By stratifying patients based on their predicted response to standard of care (SOC) chemotherapy, we identified drugs that are likely more sensitive in SOC resistant ovarian tumors. Five drugs (ABT-888, BIBW2992, gefitinib, AZD6244 and lenalidomide) exhibit higher efficacy in SOC resistant ovarian tumors when multi-platform of transcriptome profiling methods were employed. Additional in vitro and clinical sample validations were carried out and verified the effectiveness of these agents. Our candidate drugs hold great potential to improve clinical outcome of chemoresistant ovarian cancer.

Identification of MicroRNAs as Breast Cancer Prognosis Markers through the Cancer Genome Atlas.

Breast cancer is the second-most common cancer and second-leading cause of cancer mortality in American women. The dysregulation of microRNAs (miRNAs) plays a key role in almost all cancers, including breast cancer. We comprehensively analyzed miRNA expression, global gene expression, and patient survival from the Cancer Genomes Atlas (TCGA) to identify clinically relevant miRNAs and their potential gene targets in breast tumors. In our analysis, we found that increased expression of 12 mature miRNAs-hsa-miR-320a, hsa-miR-361-5p, hsa-miR-103a-3p, hsa-miR-21-5p, hsa-miR-374b-5p, hsa-miR-140-3p, hsa-miR-25-3p, hsa-miR-651-5p, hsa-miR-200c-3p, hsa-miR-30a-5p, hsa-miR-30c-5p, and hsa-let-7i-5p -each predicted improved breast cancer survival. Of the 12 miRNAs, miR-320a, miR-361-5p, miR-21-5p, miR-103a-3p were selected for further analysis. By correlating global gene expression with miRNA expression and then employing miRNA target prediction analysis, we suggest that the four miRNAs may exert protective phenotypes by targeting breast oncogenes that contribute to patient survival. We propose that miR-320a targets the survival-associated genes RAD51, RRP1B, and TDG; miR-361-5p targets ARCN1; and miR-21-5p targets MSH2, RMND5A, STAG2, and UBE2D3. The results of our stringent bioinformatics approach for identifying clinically relevant miRNAs and their targets indicate that miR-320a, miR-361-5p, and miR-21-5p may contribute to breast cancer survival.

High Expression of miR-532-5p, a Tumor Suppressor, Leads to Better Prognosis in Ovarian Cancer Both In Vivo and In Vitro.

Ovarian cancer is the leading cause of death for gynecologic cancers, ranking fifth overall for cancer-related death among women. The identification of biomarkers and the elucidation of molecular mechanisms for improving treatment options have received extensive efforts in ovarian cancer research. miRNAs have high potential to act as both ovarian cancer biomarkers and as critical regulators of ovarian tumor behavior. We comprehensively analyzed global mRNA, miRNA expression, and survival data for ovarian cancer from The Cancer Genome Atlas (TCGA) to pinpoint miRNAs that play critical roles in ovarian cancer survival through their effect on mRNA expression. We performed miRNA overexpression and gene knockdown experiments to confirm mechanisms predicted in our bioinformatics approach. We established that overexpression of miR-532-5p in OVCAR-3 cells resulted in a significant decrease in cell viability over a 96-hour time period. In the TCGA ovarian cancer dataset, we found 67 genes whose expression levels were negatively correlated with miR-532-5p expression and correlated with patient survival, such as WNT9A, CSNK2A2, CHD4, and SH3PXD2A The potential miR-532-5p-regulated gene targets were found to be enriched in the Wnt pathway. Overexpression of miR-532-5p through miRNA mimic caused downregulation of CSNK2A2, CHD4, and SH3PXD2A in the OVCAR-3 cell line. We have discovered and validated the tumor-suppressing capabilities of miR-532-5p both in vivo through TCGA analysis and in vitro through ovarian cancer cell lines. Our work highlights the potential clinical importance of miR-532-5p expression in ovarian cancer patients. Mol Cancer Ther; 15(5); 1123-31. ©2016 AACR.

The impact of the Cancer Genome Atlas on lung cancer.

The Cancer Genome Atlas (TCGA) has profiled more than 10,000 samples derived from 33 types of cancer to date, with the goal of improving our understanding of the molecular basis of cancer and advancing our ability to diagnose, treat, and prevent cancer. This review focuses on lung cancer as it is the leading cause of cancer-related mortality worldwide in both men and women. Particularly, non-small cell lung cancers (including lung adenocarcinoma and lung squamous cell carcinoma) were evaluated. Our goal was to demonstrate the impact of TCGA on lung cancer research under 4 themes: diagnostic markers, disease progression markers, novel therapeutic targets, and novel tools. Examples are given related to DNA mutation, copy number variation, messenger RNA, and microRNA expression along with methylation profiling.