Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function.

CHD7 is one of nine members of the chromodomain helicase DNA–binding domain family of ATP–dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP–Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP–seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7 +/+, Chd7 +/−, and Chd7 −/− ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES–specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.

Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

). Whereas these five residues were quantitatively modified, Arg 303 was asymmetrically dimethylated in <33% of hnRNP K and Arg 287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNAbinding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.Arginine dimethylation is a common post-translational modification in eukaryotes (1-5). The enzymes responsible for this modification are the protein-arginine methyltransferases (PRMTs).3 They are classified in two groups (2). Type I enzymes promote the formation of asymmetricThe known mammalian type I enzymes are PRMT1, PRMT2, PRMT3, PRMT4/coactivator-associated arginine methyltransferase 1, PRMT6, and the recently discovered brain-specific PRMT8 (6 -11). Type II enzymes catalyze the symmetric N G ,NЈ G -dimethylation of arginine residues. PRMT5 and PRMT7 are the mammalian type II enzymes described so far (12)(13)(14). PRMT1, which is predominantly localized to the cytoplasm (15), is thought to account for the generation of ϳ85% of asymmetric dimethylarginine residues (16) Many proteins involved in RNA metabolism like hnRNP A1 (36), hnRNP A2 (37), Sam68 (38), and SAF-A (hnRNP U) (39) contain regions with clustered arginine residues in Arg-Gly-Gly motifs (RGG box) or RG repeats. These arginine residues are typically asymmetrically dimethylated. In addition, asymmetric arginine dimethylation has also been found in clustered RXR motifs (40) and other sequences. Therefore, the prediction of a methylated arginine is difficult. Furthermore, the enzyme responsible for the dimethylation of a particular protein is unknown in many cases, and the substrate specificities of the different methyltransferases remain poorly characterized. The exact knowledge of the methylated arginine residues and their quantitative distribution as well as the identification of the relevant methyltransferases is a prerequisite for the functional analysis of arginine methylation.HnRNP K belongs to the family of heterogeneous nuclear RNPs that participate in the processing of pre-mRNAs and in the export of mRNAs from the nucleus. An N-terminal bipartite nuclear-localization signal and an hnRNP K-specific nuclear shuttling signal confer the capacity for bi-directional transport across the nuclear envelop (41, 42). The cytoplasmic accumulation of hnRNP K is mediated by its Erk-dependent serine phosphorylation (43). In the cytoplasm hnRNP K functions in the post-tr...