Genomes are organized into high-level 3-dimensional structures, and DNA elements separated by long genomic distances could functionally interact. Many transcription factors bind to regulatory DNA elements distant from gene promoters. While distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Therefore, we developed Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) for de novo detection of global chromatin interactions, and comprehensively mapped the chromatin interaction network bound by oestrogen receptor α (ERα) in the human genome. We found that most high-confidence remote ERα binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ERα functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.

CorrectionsMEDICAL SCIENCES. For the article ''Initiating oncogenic event determines gene-expression patterns of human breast cancer models,''

IntroductionWe developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer.MethodsThrough microarray informatics, Cox proportional hazards regression was used to analyze the correlation between gene expression and distant metastasis-free survival (DMFS) of patients in 14 independent breast cancer cohorts. JMJD6 emerged as a top candidate gene robustly associated with poor patient survival. Immunohistochemistry, siRNA-mediated silencing, and forced overexpression of JMJD6 in cell-based assays elucidated molecular mechanisms of JMJD6 action in breast cancer progression and shed light on the clinical breast cancer subtypes relevant to JMJD6 action.ResultsJMJD6 was expressed at highest levels in tumors associated with worse outcomes, including ER- and basal-like, Claudin-low, Her2-enriched, and ER+ Luminal B tumors. High nuclear JMJD6 protein was associated with ER negativity, advanced grade, and poor differentiation in tissue microarrays. Separation of ER+/LN- patients that received endocrine monotherapy indicated that JMJD6 is predictive of poor outcome in treatment-specific subgroups. In breast cancer cell lines, loss of JMJD6 consistently resulted in suppressed proliferation but not apoptosis, whereas forced stable overexpression increased growth. In addition, knockdown of JMJD6 in invasive cell lines, such as MDA-MB231, decreased motility and invasion, whereas overexpression in MCF-7 cells slightly promoted motility but did not confer invasive growth. Microarray analysis showed that the most significant transcriptional changes occurred in cell-proliferation genes and genes of the TGF-β tumor-suppressor pathway. High proliferation was characterized by constitutively high cyclin E protein levels. The inverse relation of JMJD6 expression with TGF-β2 could be extrapolated to the breast cancer cohorts, suggesting that JMJD6 may affect similar pathways in primary breast cancer.ConclusionsJMJD6 is a novel biomarker of tumor aggressiveness with functional implications in breast cancer growth and migration.

BackgroundGastric cancer is the second highest cause of global cancer mortality. To explore the complete repertoire of somatic alterations in gastric cancer, we combined massively parallel short read and DNA paired-end tag sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal instability and the other with microsatellite instability.ResultsIntegrative analysis and de novo assemblies revealed the architecture of a wild-type KRAS amplification, a common driver event in gastric cancer. We discovered three distinct mutational signatures in gastric cancer - against a genome-wide backdrop of oxidative and microsatellite instability-related mutational signatures, we identified the first exome-specific mutational signature. Further characterization of the impact of these signatures by combining sequencing data from 40 complete gastric cancer exomes and targeted screening of an additional 94 independent gastric tumors uncovered ACVR2A, RPL22 and LMAN1 as recurrently mutated genes in microsatellite instability-positive gastric cancer and PAPPA as a recurrently mutated gene in TP53 wild-type gastric cancer.ConclusionsThese results highlight how whole-genome cancer sequencing can uncover information relevant to tissue-specific carcinogenesis that would otherwise be missed from exome-sequencing data.

Breast cancers with positive expression of Estrogen Receptor (ER+) are treated with anti-hormone/endocrine therapy which targets the activity of the receptor, the half-life of the receptor or the availability of estrogen. This has significantly decreased mortality in women with ER+ breast cancer, however, about 25–30% of treated women run the risk or recurrence due to either intrinsic or acquired resistance to endocrine therapies. While ER itself is a predictor of response to such therapies, there exists a need to find more biomarkers and novel targets to treat resistant tumors. In this review, we summarize the known mechanisms and describe the ability of genomics in unraveling rare mutations and gene rearrangements that may impact the development of resistance and therefore treatment of ER+ breast cancer in the near future.

The goal of this study was to develop a sensitive, simple, and widely applicable assay to measure copy numbers of specific mRNAs using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and identify a profile of gene expression closely associated with angiogenesis. We measured a panel of nine potential angiogenesis markers from a mouse transgenic model of prostate adenocarcinoma (TRAMP) and a mouse skin model of vascular endothelial growth factor (VEGF)-driven angiogenesis. In both models, expression of VEGF correlated with expression of mRNAs encoding other angiogenic cytokines (angiopoietin-1 and angiopoietin-2), endothelial cell receptor tyrosine kinases (Flt-1, KDR, Tie-1), and endothelial cell adhesion molecules (VE-cadherin, PECAM-1). Relative to control, in dermis highly stimulated by VEGF, the Ang-2 mRNA transcript numbers increased 35-fold, PECAM-1 and VE-cadherin increased 10-fold, Tie-1 increased 8-fold, KDR and Flt-1 each increased 4-fold, and Ang-1 increased 2-fold. All transcript numbers were correspondingly reduced in skin with less VEGF expression, indicating a relationship of each of these seven markers with VEGF. Thus, this study identifies a highly efficient method for precise quantification of a panel of seven specific mRNAs that correlate with VEGF expression and VEGF-induced neovascularization, and it provides evidence that real-time quantitative RT-PCR offers a highly sensitive strategy for monitoring angiogenesis. Angiogenesis, the growth of new blood vessels, is widely regarded as an attractive target for controlling cancer and other important pathologies including retinopathy. Angiogenesis occurs early in tumor progression.1,2 Moreover, angiogenesis correlates not only with the onset of tumor development but also with growth and invasion of established tumors.1,3 Consequently, tools for quantifying angiogenesis are valuable for molecular profiling of tumors, monitoring the status of tumor progression and estimating the malignant potential of established tumors. In addition, molecular quantification of angiogenesis would be useful for the study of a variety of experimental models of pathological angiogenesis including retinopathy and arthritis.The most widely used technique to assess angiogenesis in clinical settings relies on immunohistochemical staining of blood vessels in fixed biopsies of specimens. Even with the use of sophisticated computerized imaging systems, this technique is labor-intensive and cannot be used as a quick and quantitative screening procedure. Individual vascular markers have been studied by quantitative and semi-quantitative techniques usually in a single tissue, but there has been little work on a comprehensive assay suitable for a varying degrees of angiogenesis. Thus, there is a need to develop a sensitive molecular assay, that allows molecular profiling of new vessel growth and that can be used to monitor angiogenesis and the efficacy of chemopreventive and other drug-based therapies toward suppressing angiogenesis.Real-time quan...

A dramatic reduction in the expression of a novel phospholipid hydroperoxide glutathione peroxidase (PHGPx), which incorporates cysteine instead of selenocysteine in the conserved catalytic motif was observed in a microarray analysis using cDNAs amplified from mRNA of Brca1-null mouse embryonic fibroblasts. This non-selenocysteine PHGPx named NPGPx is a cytoplasmic protein with molecular mass of ϳ22 kDa and has little detectable glutathione peroxidase activity in vitro. Ectopic expression of NPGPx in Brca1-null cells that were sensitive to oxidative stress induced by hydrogen peroxide conferred a similar resistance level to that of the wild-type cells, suggesting the importance of this protein in reducing oxidative stress. Expression of NPGPx was found in many tissues, including developing mammary gland. However, the majority of breast cancer cell lines studied (11 of 12) expressed very low or undetectable levels of NPGPx irrespective of BRCA1 status. Re-expression of NPGPx in breast cancer lines, MCF-7 and HCC1937, which have very little or no endogenous NPGPx, induced resistance to eicosapentaenoic acid (an omega-3 type of polyunsaturated fatty acid)-mediated cell death. Conversely, inhibition of the expression of NPGPx by the specific small interfering RNA in HS578T breast cancer cells that originally express substantial amounts of endogenous NPGPx increased their sensitivity to eicosapentaenoic acid-mediated cell death. Thus, NPGPx plays an essential role in breast cancer cells in alleviating oxidative stress generated from polyunsaturated fatty acid metabolism.

Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB). Here, we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. γ-H2AX-enriched regions of endogenous origin in replicating cells included sub-telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin. γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Sub-telomeres were less responsive to external DNA damage than to endogenous stress. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.