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We used mRNA tagging to identify genes expressed in the intestine of C. elegans. Animals expressing an epitope-tagged protein that binds the poly-A tail of mRNAs (FLAG::PAB-1) from an intestine-specific promoter (ges-1) were used to immunoprecipitate FLAG::PAB-1/mRNA complexes from the intestine. A total of 1938 intestine-expressed genes (P<0.001) were identified using DNA microarrays. First, we compared the intestine-expressed genes with those expressed in the muscle and germline, and identified 510 genes enriched in all three tissues and 624 intestine-, 230 muscle- and 1135 germ line-enriched genes. Second, we showed that the 1938 intestine-expressed genes were physically clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the commonly expressed genes showed more chromosomal clustering than the tissue-enriched genes, suggesting that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of intestinal gene expression, we searched for regulatory motifs. This analysis found that the promoters of the intestine genes were enriched for the GATA transcription factor consensus binding sequence. We experimentally verified these results by showing that the GATA motif is required in cis and that GATA transcription factors are required in trans for expression of these intestinal genes. [ABSTRACT FROM AUTHOR]

Most of the airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated basal progenitor cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia, there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in coordinating multiple cellular processes required for epithelial morphogenesis, differentiation, remodeling, and repair. However, only a few target genes have been identified, and little is known about GRHL function in the adult lung. Here we focus on the role of GRHL2 in primary human bronchial epithelial cells, both as undifferentiated progenitors and as they differentiate in air-liquid interface culture into an organized mucociliary epithelium with transepithelial resistance. Using a dominant-negative protein or shRNA to inhibit GRHL2, we follow changes in epithelial phenotype and gene transcription using RNA sequencing or microarray analysis. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2 in both undifferentiated cells and air-liquid interface cultures. Using ChIP sequencing to map sites of GRHL2 binding in the basal cells, we identify 7,687 potential primary targets and confirm that GRHL2 binding is strongly enriched near GRHL2-regulated genes. Taken together, the results support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell morphogenesis, adhesion, and motility. [ABSTRACT FROM AUTHOR]

Summary: Th17 cells have critical roles in mucosal defense and are major contributors to inflammatory disease. Their differentiation requires the nuclear hormone receptor RORγt working with multiple other essential transcription factors (TFs). We have used an iterative systems approach, combining genome-wide TF occupancy, expression profiling of TF mutants, and expression time series to delineate the Th17 global transcriptional regulatory network. We find that cooperatively bound BATF and IRF4 contribute to initial chromatin accessibility and, with STAT3, initiate a transcriptional program that is then globally tuned by the lineage-specifying TF RORγt, which plays a focal deterministic role at key loci. Integration of multiple data sets allowed inference of an accurate predictive model that we computationally and experimentally validated, identifying multiple new Th17 regulators, including Fosl2, a key determinant of cellular plasticity. This interconnected network can be used to investigate new therapeutic approaches to manipulate Th17 functions in the setting of inflammatory disease. [ABSTRACT FROM AUTHOR]

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. [ABSTRACT FROM AUTHOR]

CTCF is a ubiquitously expressed regulator of fundamental genomic processes including transcription, intra- and interchromosomal interactions, and chromatin structure. Because of its critical role in genome function, CTCF binding patterns have long been assumed to be largely invariant across different cellular environments. Here we analyze genome- wide occupancy patterns of CTCF by ChIP-seq in 19 diverse human cell types, including normal primary cells and immortal lines. We observed highly reproducible yet surprisingly plastic genomic binding landscapes, indicative of strong cell-selective regulation of CTCF occupancy. Comparison with massively parallel bisulfite sequencing data indicates that 41% of variable CTCF binding is linked to differential DNA methylation, concentrated at two critical positions within the CTCF recognition sequence. Unexpectedly, CTCF binding patterns were markedly different in normal versus immortal cells, with the latter showing widespread disruption of CTCF binding associated with increased methylation. Strikingly, this disruption is accompanied by up-regulation of CTCF expression, with the result that both normal and immortal cells maintain the same average number of CTCF occupancy sites genome-wide. These results reveal a tight linkage between DNA methylation and the global occupancy patterns of a major sequence-specific regulatory factor. [ABSTRACT FROM AUTHOR]

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode. org/) portals. [ABSTRACT FROM AUTHOR]

A complex interplay between transcription factors (TFs) and the genome regulates transcription. However, connecting variation in genome sequence with variation in TF binding and gene expression is challenging due to environmental differences between individuals and cell types. To address this problem, we measured genome-wide differential allelic occupancy of 24 TFs and EP300 in a human lymphoblastoid cell line GM12878. Overall, 5% of human TF binding sites have an allelic imbalance in occupancy. At many sites, TFs clustered in TF-binding hubs on the same homolog in especially open chromatin. While genetic variation in core TF binding motifs generally resulted in large allelic differences in TF occupancy, most allelic differences in occupancy were subtle and associated with disruption of weak or noncanonical motifs. We also measured genome-wide differential allelic expression of genes with and without heterozygous exonic variants in the same cells. We found that genes with differential allelic expression were overall less expressed both in GM12878 cells and in unrelated human cell lines. Comparing TF occupancy with expression, we found strong association between allelic occupancy and expression within 100 bp of transcription start sites (TSSs), and weak association up to 100 kb from TSSs. Sites of differential allelic occupancy were significantly enriched for variants associated with disease, particularly autoimmune disease, suggesting that allelic differences in TF occupancy give functional insights into intergenic variants associated with disease. Our results have the potential to increase the power and interpretability of association studies by targeting functional intergenic variants in addition to protein coding sequences. [ABSTRACT FROM AUTHOR]

Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdha, -ß, and -y). This diversity is generated by a combination of stochastic promoter choice and alternative premRNA splicing. Here we show that both the insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two highly conserved DNA sequences, the first within and the second downstream of transcriptionally active Pcdha promoters. Both CTCF and Rad21 bind to these sites in vitro and in vivo, this binding directly correlates with alternative isoform expression, and knocking down CTCF expression reduces alternative isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding sites was identified within a distant enhancer element (HS5-1), which is required for normal levels of alternative isoform expression. We also identify an additional, unique regulatory role for cohesin, as Rad21 binds to another enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression of the constitutive, biallelically expressed Pcdha isoforms ad and ac2. We propose that CTCF and the cohesin complex initiate and maintain Pcdha promoter choice by mediating interactions between Pcdha promoters and enhancers. [ABSTRACT FROM AUTHOR]

By disrupting microRNA (miRNA) biogenesis, we previously showed that this pathway is critical for the differentiation and function of T cells. Although various cloning studies have shown that many miRNAs are expressed during T cell development, and in a dynamic manner, it was unclear how comprehensive these earlier analyses were. We therefore decided to profile miRNA expression by next generation sequencing. Furthermore, we profiled miRNA expression starting from the hematopoietic stem cell. This analysis revealed that miRNA expression during T cell development is extremely dynamic, with 645 miRNAs sequenced, and the expression of some varying by as much as 3 orders of magnitude. Furthermore, changes in precursor processing led to altered mature miRNA sequences. We also analyzed the structures of the primary miRNA transcripts expressed in T cells and found that many were extremely long. The longest was pri-mir-29b-1/29a at ∼168 kb. All the long pri-miRNAs also displayed extensive splicing. Our findings indicate that miRNA expression during T cell development is both a highly dynamic and a highly regulated process. [ABSTRACT FROM AUTHOR]

The methylation of cytosines in CpG dinucleotides is essential for cellular differentiation and the progression of many cancers, and it plays an important role in gametic imprinting. To assess variation and inheritance of genome-wide patterns of DNA methylation simultaneously in humans, we applied reduced representation bisulfite sequencing (RRBS) to somatic DNA from six members of a three-generation family. We observed that 8.1% of heterozygous SNPs are associated with differential methylation in cis, which provides a robust signature for Mendelian transmission and relatedness. The vast majority of differential methylation between homologous chromosomes (>92%) occurs on a particular haplotype as opposed to being associated with the gender of the parent of origin, indicating that genotype affects DNA methylation of far more loci than does gametic imprinting. We found that 75% of genotype-dependent differential methylation events in the family are also seen in unrelated individuals and that overall genotype can explain 80% of the variation in DNA methylation. These events are under-represented in CpG islands, enriched in intergenic regions, and located in regions of low evolutionary conservation. Even though they are generally not in functionally constrained regions, 22% (twice as many as expected by chance) of genes harboring genotype-dependent DNA methylation exhibited allele-specific gene expression as measured by RNA-seq of a lymphoblastoid cell line, indicating that some of these events are associated with gene expression differences. Overall, our results demonstrate that the influence of genotype on patterns of DNA methylation is widespread in the genome and greatly exceeds the influence of imprinting on genome-wide methylation patterns. [ABSTRACT FROM AUTHOR]