A re-annotation pipeline for Illumina BeadArrays: improving the interpretation of gene expression data.

Illumina BeadArrays are among the most popular and reliable platforms for gene expression profiling. However, little external scrutiny has been given to the design, selection and annotation of BeadArray probes, which is a fundamental issue in data quality and interpretation. Here we present a pipeline for the complete genomic and transcriptomic re-annotation of Illumina probe sequences, also applicable to other platforms, with its output available through a Web interface and incorporated into Bioconductor packages. We have identified several problems with the design of individual probes and we show the benefits of probe re-annotation on the analysis of BeadArray gene expression data sets. We discuss the importance of aspects such as probe coverage of individual transcripts, alternative messenger RNA splicing, single-nucleotide polymorphisms, repeat sequences, RNA degradation biases and probes targeting genomic regions with no known transcription. We conclude that many of the Illumina probes have unreliable original annotation and that our re-annotation allows analyses to focus on the good quality probes, which form the majority, and also to expand the scope of biological information that can be extracted

A KLF6-driven transcriptional network links lipid homeostasis and tumour growth in renal carcinoma.

Transcriptional networks are critical for the establishment of tissue-specific cellular states in health and disease, including cancer. Yet, the transcriptional circuits that control carcinogenesis remain poorly understood. Here we report that Kruppel like factor 6 (KLF6), a transcription factor of the zinc finger family, regulates lipid homeostasis in clear cell renal cell carcinoma (ccRCC). We show that KLF6 supports the expression of lipid metabolism genes and promotes the expression of PDGFB, which activates mTOR signalling and the downstream lipid metabolism regulators SREBF1 and SREBF2. KLF6 expression is driven by a robust super enhancer that integrates signals from multiple pathways, including the ccRCC-initiating VHL-HIF2A pathway. These results suggest an underlying mechanism for high mTOR activity in ccRCC cells. More generally, the link between super enhancer-driven transcriptional networks and essential metabolic pathways may provide clues to the mechanisms that maintain the stability of cell identity-defining transcriptional programmes in cancer.CRU

Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine Kras(G12D) -p53(null) , -p53(R172H) (conformational), and -p53(R270H) (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53(R270H) -specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant.This work was supported by the Medical Research Council

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53

This work was supported by the University of Cambridge; Cancer Research UK (C14303/A17197); Hutchison Whampoa. In addition, MasasN and TO were supported by the Human Frontier Science Program (RGY0078/2010); HK was supported by MEXT KAKENHI (Grant Numbers 25116005 and 26291071); KT was supported by the Japan Society for the Promotion of Science (24–8563).The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.Publisher PDFPeer reviewe

Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease

Lamins are crucial proteins for nuclear functionality. Here, we provide new evidence showing that increased lamin B1 levels contribute to the pathophysiology of Huntington's disease (HD), a CAG repeat-associated neurodegenerative disorder. Through fluorescence-activated nuclear suspension imaging, we show that nucleus from striatal medium-sized spiny and CA1 hippocampal neurons display increased lamin B1 levels, in correlation with altered nuclear morphology and nucleocytoplasmic transport disruption. Moreover, ChIP-sequencing analysis shows an alteration of lamin-associated chromatin domains in hippocampal nuclei, accompanied by changes in chromatin accessibility and transcriptional dysregulation. Supporting lamin B1 alterations as a causal role in mutant huntingtin-mediated neurodegeneration, pharmacological normalization of lamin B1 levels in the hippocampus of the R6/1 mouse model of HD by betulinic acid administration restored nuclear homeostasis and prevented motor and cognitive dysfunction. Collectively, our work points increased lamin B1 levels as a new pathogenic mechanism in HD and provides a novel target for its intervention

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.This work was supported by the University of Cambridge; Cancer Research UK (C14303/A17197); Hutchison Whampoa. In addition, MasasN and TO were supported by the Human Frontier Science Program (RGY0078/2010); HK was supported by MEXT KAKENHI (Grant Numbers 25116005 and 26291071); KT was supported by the Japan Society for the Promotion of Science (24–8563).This is the final version of the article. It first appeared at http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.100505

NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence.

Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive 'lateral induction' of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell-cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1-NOTCH-HMGA1 axis mediates the juxtacrine regulation of chromatin architecture

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer.

BACKGROUND: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. METHODS: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). RESULTS: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. CONCLUSIONS: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity

ZNF703 is a common Luminal B breast cancer oncogene that differentially regulates luminal and basal progenitors in human mammary epithelium.

The telomeric amplicon at 8p12 is common in oestrogen receptor-positive (ER+) breast cancers. Array-CGH and expression analyses of 1172 primary breast tumours revealed that ZNF703 was the single gene within the minimal amplicon and was amplified predominantly in the Luminal B subtype. Amplification was shown to correlate with increased gene and protein expression and was associated with a distinct expression signature and poor clinical outcome. ZNF703 transformed NIH 3T3 fibroblasts, behaving as a classical oncogene, and regulated proliferation in human luminal breast cancer cell lines and immortalized human mammary epithelial cells. Manipulation of ZNF703 expression in the luminal MCF7 cell line modified the effects of TGFβ on proliferation. Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony-forming cells from luminal progenitors. Taken together, these data strongly point to ZNF703 as a novel oncogene in Luminal B breast cancer

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer

Large-scale human genetic data(1-3) have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)(4-6). VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2 alpha (HIF2A) stabilization(6,7). We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele Cat rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. (8)). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.Peer reviewe