Understanding the 3D genome: Emerging impacts on human disease.

Recent burst of new technologies that allow for quantitatively delineating chromatin structure has greatly expanded our understanding of how the genome is organized in the three-dimensional (3D) space of the nucleus. It is now clear that the hierarchical organization of the eukaryotic genome critically impacts nuclear activities such as transcription, replication, as well as cellular and developmental events such as cell cycle, cell fate decision and embryonic development. In this review, we discuss new insights into how the structural features of the 3D genome hierarchy are established and maintained, how this hierarchy undergoes dynamic rearrangement during normal development and how its perturbation will lead to human disease, highlighting the accumulating evidence that links the diverse 3D genome architecture components to a multitude of human diseases and the emerging mechanisms by which 3D genome derangement causes disease phenotypes.

Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution.

The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods.

Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes.

High-throughput methods based on chromosome conformation capture have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their reliance on restriction enzymes. Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts. DNase Hi-C uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution over that of Hi-C. Coupling this method with DNA-capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 large intergenic noncoding RNA (lincRNA) promoters in two human cell lines. Our results revealed that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the Polycomb repressive complex. Our results provide the first glimpse of the cell type-specific 3D organization of lincRNA genes.

Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene.

BACKGROUND: Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (IGF2), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the IGF2 gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II. RESULTS: Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed IGF2BP1 gene. In vitro binding assays using the CTCF target motif at the IGF2BP1 gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic IGF2BP1 expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express IGF2BP1 primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of IGF2BP1 gene expression. CONCLUSIONS: Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.

Targeted deletion of multiple CTCF-binding elements in the human C-MYC gene reveals a requirement for CTCF in C-MYC expression.

BACKGROUND: Insulators and domain boundaries both shield genes from adjacent enhancers and inhibit intrusion of heterochromatin into transgenes. Previous studies examined the functional mechanism of the MYC insulator element MINE and its CTCF binding sites in the context of transgenes that were randomly inserted into the genome by transfection. However, the contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene. METHODOLOGY/PRINCIPAL FINDINGS: To determine the impact of CTCF binding on MYC expression, a series of mutant human chromosomal alleles was prepared in homologous recombination-efficient DT40 cells and individually transferred by microcell fusion into murine cells. Functional tests reported here reveal that deletion of CTCF binding elements within the MINE does not impact the capacity of this locus to correctly organize an 'accessible' open chromatin domain, suggesting that these sites are not essential for the formation of a competent, transcriptionally active locus. Moreover, deletion of the CTCF site at the MYC P2 promoter reduces transcription but does not affect promoter acetylation or serum-inducible transcription. Importantly, removal of either CTCF site leads to DNA methylation of flanking sequences, thereby contributing to progressive loss of transcriptional activity. CONCLUSIONS: These findings collectively demonstrate that CTCF-binding at the human MYC locus does not repress transcriptional activity but is required for protection from DNA methylation.

CTCF physically links cohesin to chromatin.

Cohesin is required to prevent premature dissociation of sister chromatids after DNA replication. Although its role in chromatid cohesion is well established, the functional significance of cohesin's association with interphase chromatin is not clear. Using a quantitative proteomics approach, we show that the STAG1 (Scc3/SA1) subunit of cohesin interacts with the CCTC-binding factor CTCF bound to the c-myc insulator element. Both allele-specific binding of CTCF and Scc3/SA1 at the imprinted IGF2/H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF-binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF. A large-scale genomic survey using ChIP-Chip demonstrates that Scc3/SA1 binding strongly correlates with the CTCF-binding site distribution in chromosomal arms. However, some chromosomal sites interact exclusively with CTCF, whereas others interact with Scc3/SA1 only. Furthermore, immunofluorescence microscopy and ChIP-Chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase. These results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion. These results have implications for the functional role of cohesin subunits in the pathogenesis of Cornelia de Lange syndrome and Roberts syndromes.

Identification of DNA methylation in 3' genomic regions that are associated with upregulation of gene expression in colorectal cancer.

Restriction landmark genomic scanning (RLGS), a method for the two-dimensional display of end-labeled DNA restriction fragments, was utilized to identify genomic regions of CpG island methylation associated with human colon cancer. An average of 1.5% of the RLGS loci/spots are lost or significantly reduced in sporadic primary colon tumors relative to normal colon mucosa from the same patient. This may represent tumor specific methylation of about 400 CpG islands in sporadic colon cancer. A number of RLGS loci exhibiting frequent loss associated with colon cancer were cloned. DNA sequence analysis indicated that the RLGS loci identified genomic regions characteristic of CpG islands. A number of methods including bisulfite genomic sequencing as well as quantitative MassARRAY methylation analysis (www.sequenom.com) confirmed tumor specific methylation at several of these loci. DNA database searches indicated that candidate genes associated with these loci include transcription factors and genes involved in signal transduction (52%), and genes of unknown function (37%). Expression analysis using quantitative real time RT-PCR indicates that methylation of some CpG islands located in non-promoter regions were associated with upregulation of gene expression in colorectal cancer. These results indicate that alterations in methylation status within CpG islands in colon tumors may have complex consequences on gene expression and tumorigenesis, sometimes resulting in up regulation or ectopic gene expression that may involve novel regulatory mechanisms.

Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.

Intergenic transcription is not required in Th2 cells to maintain histone acetylation and transcriptional permissiveness at the Il4-Il13 locus.

Noncoding RNA transcripts mapping to intergenic regions of the Il4-Il13 locus have been detected in Th2 cells harboring transcriptionally permissive Il4 and Il13 genes but not in Th1 cells where these genes are repressed. This correlation has given rise to the idea that intergenic transcription may be involved in maintaining the "open" chromatin structure of the Il4-Il13 locus in Th2 cells. We present evidence from real-time RT-PCR, nuclear run on, chromatin immunoprecipitation and 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside-mediated transcriptional inhibition analyses that argue against this hypothesis. Instead, our results are consistent with an alternative role for intergenic transcription in the maintenance of transcriptional silence in Th1-primed cells.

Transcription-induced chromatin remodeling at the c-myc gene involves the local exchange of histone H2A.Z.

The post-translational modification of histones and the incorporation of core histone variants play key roles in governing gene expression. Many eukaryotic genes regulate their expression by limiting the escape of RNA polymerase from promoter-proximal pause sites. Here we report that elongating RNA polymerase II complexes encounter distinct chromatin landscapes that are marked by methylation of lysine residues Lys(4), Lys(79), and Lys(36) of histone H3. However, neither histone methylation nor acetylation directly regulates the release of elongation complexes stalled at promoter-proximal pause sites of the c-myc gene. In contrast, transcriptional activation is associated with local displacement of the histone variant H2A.Z within the transcribed region and incorporation of the major histone variant H2A. This result indicates that transcribing RNA polymerase II remodels chromatin in part through coincident displacement of H2A.Z-H2B dimers and incorporation of H2A-H2B dimers. In combination, these results suggest a new model in which the incorporation of H2A.Z into nucleosomes down-regulates transcription; at the same time it may act as a cellular memory for transcriptionally poised gene domains.

A permissive role for phosphatidylinositol 3-kinase in the Stat5-mediated expression of cyclin D2 by the interleukin-2 receptor.

The interleukin-2 (IL-2) receptor promotes T cell proliferation in part by inducing the expression of D-type cyclins, which enable cells to progress from the G1 to S phase of the cell cycle. We previously showed that the IL-2 receptor induces expression of cyclin D2 by activating the transcription factor Stat5, which binds directly and immediately to a site upstream of the cyclin D2 promoter. We show here that subsequent transcription of the cyclin D2 gene occurs by a delayed, cycloheximide-sensitive mechanism, which implies the involvement of additional regulatory mechanisms. The transcription factor c-Myc is induced by Stat5 and is reported to bind to two E box motifs in the cyclin D2 promoter. However, in IL-2-stimulated T cells, c-Myc does not appear to be involved in cyclin D2 induction, since we found that these two E boxes are preferentially bound by USF-1 and USF-2 and, moreover, are dispensable for cyclin D2 promoter activity. Instead, we found that Stat5 activates the phosphatidylinositol 3-kinase (PI3 kinase) pathway by a delayed, cycloheximide-sensitive mechanism and that PI3 kinase activity is essential for the induction of cyclin D2 by Stat5. Chromatin immunoprecipitation experiments revealed that PI3 kinase is required for the optimal binding of RNA polymerase II to the promoters of cyclin D2 as well as other genes. Our results reveal a novel link between PI3 kinase and RNA polymerase II promoter binding activity and demonstrate discrete, coordinated roles for the PI3 kinase and Stat5 pathways in cyclin D2 transcription.

The c-myc insulator element and matrix attachment regions define the c-myc chromosomal domain.

Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.

An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.