Publisher Correction: ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters.

This corrects the article DOI: 10.1038/ncomms7186.

Publisher Correction: Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

This corrects the article DOI: 10.1038/ncomms5999.

ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters.

Chromatin interactions connect distal regulatory elements to target gene promoters guiding stimulus- and lineage-specific transcription. Few factors securing chromatin interactions have so far been identified. Here, by integrating chromatin interaction maps with the large collection of transcription factor-binding profiles provided by the ENCODE project, we demonstrate that the zinc-finger protein ZNF143 preferentially occupies anchors of chromatin interactions connecting promoters with distal regulatory elements. It binds directly to promoters and associates with lineage-specific chromatin interactions and gene expression. Silencing ZNF143 or modulating its DNA-binding affinity using single-nucleotide polymorphisms (SNPs) as a surrogate of site-directed mutagenesis reveals the sequence dependency of chromatin interactions at gene promoters. We also find that chromatin interactions alone do not regulate gene expression. Together, our results identify ZNF143 as a novel chromatin-looping factor that contributes to the architectural foundation of the genome by providing sequence specificity at promoters connected with distal regulatory elements.

Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology.

Chromatin profiling reveals regulatory network shifts and a protective role for hepatocyte nuclear factor 4α during colitis.

Transcriptional regulatory mechanisms likely contribute to the etiology of inflammatory bowel disease (IBD), as genetic variants associated with the disease are disproportionately found at regulatory elements. However, the transcription factors regulating colonic inflammation are unclear. To identify these transcription factors, we mapped epigenomic changes in the colonic epithelium upon inflammation. Epigenetic marks at transcriptional regulatory elements responded dynamically to inflammation and indicated a shift in epithelial transcriptional factor networks. Active enhancer chromatin structure at regulatory regions bound by the transcription factor hepatocyte nuclear factor 4α (HNF4A) was reduced during colitis. In agreement, upon an inflammatory stimulus, HNF4A was downregulated and showed a reduced ability to bind chromatin. Genetic variants that confer a predisposition to IBD map to HNF4A binding sites in the human colon cell line CaCo2, suggesting impaired HNF4A binding could underlie genetic susceptibility to IBD. Despite reduced HNF4A binding during inflammation, a temporal knockout model revealed HNF4A still actively protects against inflammatory phenotypes and promotes immune regulatory gene expression in the inflamed colonic epithelium. These findings highlight the potential for HNF4A agonists as IBD therapeutics.

Intratumor DNA methylation heterogeneity reflects clonal evolution in aggressive prostate cancer.

Despite much evidence on epigenetic abnormalities in cancer, it is currently unclear to what extent epigenetic alterations can be associated with tumors' clonal genetic origins. Here, we show that the prostate intratumor heterogeneity in DNA methylation and copy-number patterns can be explained by a unified evolutionary process. By assaying multiple topographically distinct tumor sites, premalignant lesions, and lymph node metastases within five cases of prostate cancer, we demonstrate that both DNA methylation and copy-number heterogeneity consistently reflect the life history of the tumors. Furthermore, we show cases of genetic or epigenetic convergent evolution and highlight the diversity in the evolutionary origins and aberration spectrum between tumor and metastatic subclones. Importantly, DNA methylation can complement genetic data by serving as a proxy for activity at regulatory domains, as we show through identification of high epigenetic heterogeneity at androgen-receptor-bound enhancers. Epigenome variation thereby expands on the current genome-centric view on tumor heterogeneity.

Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits.

DNA variants (SNPs) that predispose to common traits often localize within noncoding regulatory elements such as enhancers. Moreover, loci identified by genome-wide association studies (GWAS) often contain multiple SNPs in linkage disequilibrium (LD), any of which may be causal. Thus, determining the effect of these multiple variant SNPs on target transcript levels has been a major challenge. Here, we provide evidence that for six common autoimmune disorders (rheumatoid arthritis, Crohn's disease, celiac disease, multiple sclerosis, lupus, and ulcerative colitis), the GWAS association arises from multiple polymorphisms in LD that map to clusters of enhancer elements active in the same cell type. This finding suggests a "multiple enhancer variant" hypothesis for common traits, where several variants in LD impact multiple enhancers and cooperatively affect gene expression. Using a novel method to delineate enhancer-gene interactions, we show that multiple enhancer variants within a given locus typically target the same gene. Using available data from HapMap and B lymphoblasts as a model system, we provide evidence at numerous loci that multiple enhancer variants cooperatively contribute to altered expression of their gene targets. The effects on target transcript levels tend to be modest and can be either gain- or loss-of-function. Additionally, the genes associated with multiple enhancer variants encode proteins that are often functionally related and enriched in common pathways. Overall, the multiple enhancer variant hypothesis offers a new paradigm by which noncoding variants can confer susceptibility to common traits.

Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression.

Genome-wide association studies (GWAS) have identified thousands of SNPs that are associated with human traits and diseases. But, because the vast majority of these SNPs are located in non-coding regions of the genome, the mechanisms by which they promote disease risk have remained elusive. Employing a new methodology that combines cistromics, epigenomics and genotype imputation, we annotate the non-coding regions of the genome in breast cancer cells and systematically identify the functional nature of SNPs associated with breast cancer risk. Our results show that breast cancer risk-associated SNPs are enriched in the cistromes of FOXA1 and ESR1 and the epigenome of histone H3 lysine 4 monomethylation (H3K4me1) in a cancer- and cell type-specific manner. Furthermore, the majority of the risk-associated SNPs modulate the affinity of chromatin for FOXA1 at distal regulatory elements, thereby resulting in allele-specific gene expression, which is exemplified by the effect of the rs4784227 SNP on the TOX3 gene within the 16q12.1 risk locus.

Integrative functional genomics identifies an enhancer looping to the SOX9 gene disrupted by the 17q24.3 prostate cancer risk locus.

Genome-wide association studies (GWAS) are identifying genetic predisposition to various diseases. The 17q24.3 locus harbors the single nucleotide polymorphism (SNP) rs1859962 that is statistically associated with prostate cancer (PCa). It defines a 130-kb linkage disequilibrium (LD) block that lies in an ∼2-Mb gene desert area. The functional biology driving the risk associated with this LD block is unknown. Here, we integrate genome-wide chromatin landscape data sets, namely, epigenomes and chromatin openness from diverse cell types. This identifies a PCa-specific enhancer within the rs1859962 risk LD block that establishes a 1-Mb chromatin loop with the SOX9 gene. The rs8072254 and rs1859961 SNPs mapping to this enhancer impose allele-specific gene expression. The variant allele of rs8072254 facilitates androgen receptor (AR) binding driving increased enhancer activity. The variant allele of rs1859961 decreases FOXA1 binding while increasing AP-1 binding. The latter is key to imposing allele-specific gene expression. The rs8072254 variant in strong LD with the rs1859962 risk SNP can account for the risk associated with this locus, while rs1859961 is a rare variant less likely to contribute to the risk associated with this LD block. Together, our results demonstrate that multiple genetic variants mapping to a unique enhancer looping to the SOX9 oncogene can account for the risk associated with the PCa 17q24.3 locus. Allele-specific recruitment of the transcription factors androgen receptor (AR) and activating protein-1 (AP-1) account for the increased enhancer activity ascribed to this PCa-risk LD block. This further supports the notion that an integrative genomics approach can identify the functional biology disrupted by genetic risk variants.

Epigenomic enhancer profiling defines a signature of colon cancer.

Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, even though distal regulatory elements play a central role in controlling transcription patterns. We used the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome-wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis.

Systems genetics for drug target discovery.

The collection and analysis of genomic data has the potential to reveal novel druggable targets by providing insight into the genetic basis of disease. However, the number of drugs targeting new molecular entities, approved by the US Food and Drug Administration has not increased in the years since the collection of genomic data has become commonplace. The paucity of translatable results can be partly attributed to conventional analysis methods that test one gene at a time in an effort to identify disease-associated factors as candidate drug targets. By disengaging genetic factors from their position within the genetic regulatory system, much of the information stored within the genomic data set is lost. Here we discuss how genomic data is used to identify disease-associated genes or genomic regions, how disease-associated regions are validated as functional targets, and the role network analysis can play in bridging the gap between data generation and effective drug target identification.

Layers of epistasis: genome-wide regulatory networks and network approaches to genome-wide association studies.

The conceptual foundation of the genome-wide association study (GWAS) has advanced unchecked since its conception. A revision might seem premature as the potential of GWAS has not been fully realized. Multiple technical and practical limitations need to be overcome before GWAS can be fairly criticized. But with the completion of hundreds of studies and a deeper understanding of the genetic architecture of disease, warnings are being raised. The results compiled to date indicate that risk-associated variants lie predominantly in noncoding regions of the genome. Additionally, alternative methodologies are uncovering large and heterogeneous sets of rare variants underlying disease. The fear is that, even in its fulfillment, the current GWAS paradigm might be incapable of dissecting all kinds of phenotypes. In the following text, we review several initiatives that aim to overcome these limitations. The overarching theme of these studies is the inclusion of biological knowledge to both the analysis and interpretation of genotyping data. GWAS is uninformed of biology by design and although there is some virtue in its simplicity, it is also its most conspicuous deficiency. We propose a framework in which to integrate these novel approaches, both empirical and theoretical, in the form of a genome-wide regulatory network (GWRN). By processing experimental data into networks, emerging data types based on chromatin immunoprecipitation are made computationally tractable. This will give GWAS re-analysis efforts the most current and relevant substrates, and root them firmly on our knowledge of human disease.

microRNAs reveal the interrelationships of hagfish, lampreys, and gnathostomes and the nature of the ancestral vertebrate.

Hagfish and lampreys are the only living representatives of the jawless vertebrates (agnathans), and compared with jawed vertebrates (gnathostomes), they provide insight into the embryology, genomics, and body plan of the ancestral vertebrate. However, this insight has been obscured by controversy over their interrelationships. Morphological cladistic analyses have identified lampreys and gnathostomes as closest relatives, whereas molecular phylogenetic studies recover a monophyletic Cyclostomata (hagfish and lampreys as closest relatives). Here, we show through deep sequencing of small RNA libraries, coupled with genomic surveys, that Cyclostomata is monophyletic: hagfish and lampreys share 4 unique microRNA families, 15 unique paralogues of more primitive microRNA families, and 22 unique substitutions to the mature gene products. Reanalysis of morphological data reveals that support for cyclostome paraphyly was based largely on incorrect character coding, and a revised dataset is not decisive on the mono- vs. paraphyly of cyclostomes. Furthermore, we show fundamental conservation of microRNA expression patterns among lamprey, hagfish, and gnathostome organs, implying that the role of microRNAs within specific organs is coincident with their appearance within the genome and is conserved through time. Together, these data support the monophyly of cyclostomes and suggest that the last common ancestor of all living vertebrates was a more complex organism than conventionally accepted by comparative morphologists and developmental biologists.

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.

With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.

MicroRNAs and regeneration: Let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt.

MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.