Chromatin Interaction Analysis Research Articles

Advances in Genomic Profiling and Analysis of 3D Chromatin Structure and Interaction.

Recent sequence-based profiling technologies such as high-throughput sequencing to detect fragment nucleotide sequence (Hi-C) and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) have revolutionized the field of three-dimensional (3D) chromatin architecture. It is now recognized that human genome functions as folded 3D chromatin units and looping paradigm is the basic principle of gene regulation. To better interpret the 3D data dramatically accumulating in past five years and to gain deep biological insights, huge efforts have been made in developing novel quantitative analysis methods. However, the full understanding of genome regulation requires thorough knowledge in both genomic technologies and their related data analyses. We summarize the recent advances in genomic technologies in identifying the 3D chromatin structure and interaction, and illustrate the quantitative analysis methods to infer functional domains and chromatin interactions, and further elucidate the emerging single-cell Hi-C technique and its computational analysis, and finally discuss the future directions such as advances of 3D chromatin techniques in diseases.

In Situ Capture of Chromatin Interactions by Biotinylated dCas9

Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs inchromatin remains unknown. Here, we describe aCRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identifylocus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an invivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purificationof human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis oflocus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

Abstract 5370: Examination of DNA looping near oncogenes reveals variable patterns of epigenetic landscapes in cancer

Abstract INTRODUCTION: The compaction of chromatin in the nucleus into hierarchical three-dimensional (3D) structures plays a key role in the regulation of gene expression. Advancements in high-throughput chromatin conformation capture assays have enabled the determination of this hierarchical structure in a variety of cell states, including cancer. Of note, recent studies have implicated pathogenic alterations in 3D topology of the genome with the activation of proto-oncogenes in a multitude of cancers. METHODS: To characterize the topological changes associated with different cancer phenotypes, we examine publically available epigenetic profiles from the ENCODE Project, including 3D data from Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) from breast cancer and leukemia cell lines. Integrating the chromatin topology data with transcription factor binding, DNase accessibility, and histone modification data, we uncover variable epigenetic landscapes associated with differentially active oncogenes in these cancer cell lines. RESULTS: We provide evidence of topological variability influencing differentially expressed oncogenes and tie these observations to other epigenetic changes. In particular, we show insulated-neighborhoods mediated by CTCF looping partition segments of the genome containing active oncogenes away from repressive markings like H3K4me3. Additionally, we show that variation in topology differentially localizes H3K27ac in distal regulatory regions to promoters of oncogenes, providing a means for gene activation in non-coding regions. CONCLUSIONS: Variable patterns of chromatin topology provide a unique signature and mechanism of oncogenes for cancerous phenotypes. We use publically available data to show mechanisms of activity associated with chromatin topology variation. Citation Format: Monika Perez, Ty Johannes. Examination of DNA looping near oncogenes reveals variable patterns of epigenetic landscapes in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5370. doi:10.1158/1538-7445.AM2017-5370

Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions.

Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.

Chromatin landscapes and genetic risk for juvenile idiopathic arthritis

BackgroundThe transcriptomes of peripheral blood cells in children with juvenile idiopathic arthritis (JIA) have distinct transcriptional aberrations that suggest impairment of transcriptional regulation. To gain a better understanding of this phenomenon, we studied known JIA genetic risk loci, the majority of which are located in non-coding regions, where transcription is regulated and coordinated on a genome-wide basis. We examined human neutrophils and CD4 primary T cells to identify genes and functional elements located within those risk loci.MethodsWe analyzed RNA sequencing (RNA-Seq) data, H3K27ac and H3K4me1 chromatin immunoprecipitation-sequencing (ChIP-Seq) data, and previously published chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data to characterize the chromatin landscapes within the known JIA-associated risk loci.ResultsIn both neutrophils and primary CD4+ T cells, the majority of the JIA-associated linkage disequilibrium (LD) blocks contained H3K27ac and/or H3K4me1 marks. These LD blocks were also binding sites for a small group of transcription factors, particularly in neutrophils. Furthermore, these regions showed abundant intronic and intergenic transcription in neutrophils. In neutrophils, none of the genes that were differentially expressed between untreated patients with JIA and healthy children were located within the JIA-risk LD blocks. In CD4+ T cells, multiple genes, including HLA-DQA1, HLA-DQB2, TRAF1, and IRF1 were associated with the long-distance interacting regions within the LD regions as determined from ChIA-PET data.ConclusionsThese findings suggest that genetic risk contributes to the aberrant transcriptional control observed in JIA. Furthermore, these findings demonstrate the challenges of identifying the actual causal variants within complex genomic/chromatin landscapes.

Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding.

Complex regulatory landscapes control the pleiotropic transcriptional activities of developmental genes. For most genes, the number, location, and dynamics of their associated regulatory elements are unknown. In this work, we characterized the three-dimensional chromatin microarchitecture and regulatory landscape of 446 limb-associated gene loci in mouse using Capture-C, ChIP-seq, and RNA-seq in forelimb, hindlimb at three developmental stages, and midbrain. The fine mapping of chromatin interactions revealed a strong preference for functional genomic regions such as repressed or active domains. By combining chromatin marks and interaction peaks, we annotated more than 1000 putative limb enhancers and their associated genes. Moreover, the analysis of chromatin interactions revealed two regimes of chromatin folding, one producing interactions stable across tissues and stages and another one associated with tissue and/or stage-specific interactions. Whereas stable interactions associate strongly with CTCF/RAD21 binding, the intensity of variable interactions correlates with changes in underlying chromatin modifications, specifically at the viewpoint and at the interaction site. In conclusion, this comprehensive data set provides a resource for the characterization of hundreds of limb-associated regulatory landscapes and a framework to interpret the chromatin folding dynamics observed during embryogenesis.

Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease.

BackgroundGenome-wide association studies (GWAS) have revealed many susceptibility loci for complex genetic diseases. For most loci, the causal genes have not been identified. Currently, the identification of candidate genes is predominantly based on genes that localize close to or within identified loci. We have recently shown that 92 of the 163 inflammatory bowel disease (IBD)-loci co-localize with non-coding DNA regulatory elements (DREs). Mutations in DREs can contribute to IBD pathogenesis through dysregulation of gene expression. Consequently, genes that are regulated by these 92 DREs are to be considered as candidate genes. This study uses circular chromosome conformation capture-sequencing (4C-seq) to systematically analyze chromatin-interactions at IBD susceptibility loci that localize to regulatory DNA.ResultsUsing 4C-seq, we identify genomic regions that physically interact with the 92 DRE that were found at IBD susceptibility loci. Since the activity of regulatory elements is cell-type specific, 4C-seq was performed in monocytes, lymphocytes, and intestinal epithelial cells. Altogether, we identified 902 novel IBD candidate genes. These include genes specific for IBD-subtypes and many noteworthy genes including ATG9A and IL10RA. We show that expression of many novel candidate genes is genotype-dependent and that these genes are upregulated during intestinal inflammation in IBD. Furthermore, we identify HNF4α as a potential key upstream regulator of IBD candidate genes.ConclusionsWe reveal many novel and relevant IBD candidate genes, pathways, and regulators. Our approach complements classical candidate gene identification, links novel genes to IBD and can be applied to any existing GWAS data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1100-3) contains supplementary material, which is available to authorized users.

Deciphering Noncoding RNA and Chromatin Interactions: Multiplex Chromatin Interaction Analysis by Paired-End Tag Sequencing (mChIA-PET).

Genomic DNA is dynamically associated with protein factors and folded to form chromatin fibers. The 3-dimensional (3D) configuration of the chromatin will enable the distal genetic elements to come into close proximity, allowing transcriptional regulation. Noncoding RNA can mediate the 3D structure of chromatin. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a valuable and powerful technique in molecular biology which allows the study of unbiased, genome-wide de novo chromatin interactions with paired-end tags. Here, we describe the standard version of ChIA-PET and a Multiplex ChIA-PET version.

Advances in computational ChIA‐PET data analysis

Genome‐wide chromatin interaction analysis has become important for understanding 3D topological structure of a genome as well as for linking distal cis‐regulatory elements to their target genes. Compared to the Hi‐C method, chromatin interaction analysis by paired‐end tag sequencing (ChIA‐PET) is unique, in that one can interrogate thousands of chromatin interactions (in a genome) mediated by a specific protein of interest at high resolution and reasonable cost. However, because of the noisy nature of the data, efficient analytical tools have become necessary. Here, we review some new computational methods recently developed by us and compare them with other existing methods. Our intention is to help readers to better understand ChIA‐PET results and to guide the users on selection of the most appropriate tools for their own projects.

50 - Regulatory Mutations of FOXG1 in the Context of Topological Domains

Recent HiC proximity mapping have revealed a 3D-organization of the human genome where cis-regulatory interactions occur within megabase-sized topological associating domains (TADs) ( 1 Dixon J.R. Selvaraj S. Yue F. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012; 485: 376-380 Crossref PubMed Scopus (3953) Google Scholar ). TADs are separated by boundaries (TDB) which are enriched in Transcriptional Start Sites (TSS), housekeeping genes, binding sites for CTCF insulator factor and SINE elements ( 1 Dixon J.R. Selvaraj S. Yue F. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012; 485: 376-380 Crossref PubMed Scopus (3953) Google Scholar ). Different structural variations like deletions and translocations may interfere with the TAD organization. Translocations with breakpoints outside of TAD boundaries (the majority) will invariably interfere with TAD organization, creating fusion domains, whereas deletions may or may not, depending on position and size. Although the majority of TADs are identical in human embryonic cells (hESC) and IMR90 fibroblast, some loci display a differential pattern in the two cell lines. This is exemplified by the Forkhead box G1 (FOXG1) gene on 14q12, located within a 2 Mb sized TAD in IMR90 cells which is split into two TADs in hESC. FOXG1 encodes a winged-helix transcriptional repressor critical for early telencephalon development. Coding mutations of FOXG1 causes a congenital variant of the severe neurodevelopmental disorder Rett syndrome2. Balanced chromosomal rearrangements with breakpoints downstream of FOXG1 have been shown to cause the same phenotype as coding mutations ( 2 Alosi D. Klitten L.L. Bak M. et al. Dysregulation of FOXG1 by ring chromosome 14. Mol Cytogenet. 2015; 8: 24 Crossref PubMed Scopus (6) Google Scholar , 3 Shoichet S.A. Kunde S.A. Viertel P. et al. Haploinsufficiency of novel FOXG1B variants in a patient with severe mental retardation and microcephaly. Hum Genet. 2005; 117: 536-544 Crossref PubMed Scopus (79) Google Scholar ). Such long range position effects (LRPE) are presumably caused by the removal of cis-acting regulatory elements. The LRPE critical region at the FOXG1 locus is not defined, and intra-TAD deletions that remove the hESC-specific TAD boundary distal to FOXG1 have been suggested to exemplify enhancer adoption ( 4 Lettice L.A. Daniels S. Sweeney E. et al. Enhancer-adoption as a mechanism of human developmental disease. Hum Mutat. 2011; 32: 1492-1499 Crossref PubMed Scopus (78) Google Scholar ), with the establishment of new deleterious enhancer-target gene interactions ( 5 Ibn-Salem J. Köhler S. Love M.I. et al. Deletions of chromosomal regulatory boundaries are associated with congenital disease. Genome Biol. 2014; 15: 423 Crossref PubMed Scopus (95) Google Scholar ). Herein we have mapped two new LRPE-associated translocations by mate-pair sequencing, which we use together with additional microdeletions distal to FOXG1 to define the critical LRPE-region and the evidence for or against enhancer adoption as a major feature of the FOXG1-locus.

Analysis of Long Noncoding RNA Chromatin Interactions by Chromatin Isolation by RNA Purification (ChIRP)

Gene regulation plays a critical role in complex cellular processes such as development, differentiation, and cellular response to environmental changes. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non‐coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored.Chromatin Isolation by RNA Purification (ChIRP) is a method that allows discovery of the sites of interaction of chromatin‐associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe‐based hybridization to target RNA molecules in chromatin. DNA can be isolated from recovered chromatin and analyzed by quantitative PCR or next generation sequencing (ChIRP‐seq). Alternatively, RNA may also be isolated to detect other RNA molecules that may be associated with the RNA of interest.To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19) and peaks were called. We successfully identified several NEAT1 binding sites in the genomic DNA sequence.In summary the methods developed allow unbiased discovery of RNA‐associated genomic DNA sequences, RNA sequences, and potentially proteins.

3CDB: a manually curated database of chromosome conformation capture data.

Chromosome conformation capture (3C) is a biochemical technology to analyse contact frequencies between selected genomic sites in a cell population. Its recent genomic variants, e.g. Hi-C/ chromatin interaction analysis by paired-end tag (ChIA-PET), have enabled the study of nuclear organization at an unprecedented level. However, due to the inherent low resolution and ultrahigh cost of Hi-C/ChIA-PET, 3C is still the gold standard for determining interactions between given regulatory DNA elements, such as enhancers and promoters. Therefore, we developed a database of 3C determined functional chromatin interactions (3CDB; http://3cdb.big.ac.cn). To construct 3CDB, we searched PubMed and Google Scholar with carefully designed keyword combinations and retrieved more than 5000 articles from which we manually extracted 3319 interactions in 17 species. Moreover, we proposed a systematic evaluation scheme for data reliability and classified the interactions into four categories. Contact frequencies are not directly comparable as a result of various modified 3C protocols employed among laboratories. Our evaluation scheme provides a plausible solution to this long-standing problem in the field. A user-friendly web interface was designed to assist quick searches in 3CDB. We believe that 3CDB will provide fundamental information for experimental design and phylogenetic analysis, as well as bridge the gap between molecular and systems biologists who must now contend with noisy high-throughput data.Database URL: http://3cdb.big.ac.cn

Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization.

BackgroundThe impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activated transcription factors to recruitment of activated transcription factors to pre-established long-range interactions.ResultsUsing circular chromosome conformation capture coupled with next generation sequencing and high-resolution chromatin interaction analysis by paired-end tag sequencing of P300, we observed agonist-induced changes in long-range chromatin interactions, and uncovered interconnected enhancer–enhancer hubs spanning up to one megabase. The vast majority of activated glucocorticoid receptor and nuclear factor kappa-b appeared to join pre-existing P300 enhancer hubs without affecting the chromatin conformation. In contrast, binding of the activated transcription factors to loci with their consensus response elements led to the increased formation of an active epigenetic state of enhancers and a significant increase in long-range interactions within pre-existing enhancer networks. De novo enhancers or ligand-responsive enhancer hubs preferentially interacted with ligand-induced genes.ConclusionsWe demonstrate that, at a subset of genomic loci, ligand-mediated induction leads to active enhancer formation and an increase in long-range interactions, facilitating efficient regulation of target genes. Therefore, our data suggest an active role of signal-dependent transcription factors in chromatin and long-range interaction remodeling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0832-9) contains supplementary material, which is available to authorized users.

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer.

Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATα cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.

Chromatin interaction analysis reveals changes in small chromosome and telomere clustering between epithelial and breast cancer cells.

BackgroundHigher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines.ResultsOur studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16–22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16–22 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells.ConclusionsWe show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0768-0) contains supplementary material, which is available to authorized users.

Mango: a bias-correcting ChIA-PET analysis pipeline

Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) is an established method for detecting genome-wide looping interactions at high resolution. Current ChIA-PET analysis software packages either fail to correct for non-specific interactions due to genomic proximity or only address a fraction of the steps required for data processing. We present Mango, a complete ChIA-PET data analysis pipeline that provides statistical confidence estimates for interactions and corrects for major sources of bias including differential peak enrichment and genomic proximity. Comparison to the existing software packages, ChIA-PET Tool and ChiaSig revealed that Mango interactions exhibit much better agreement with high-resolution Hi-C data. Importantly, Mango executes all steps required for processing ChIA-PET datasets, whereas ChiaSig only completes 20% of the required steps. Application of Mango to multiple available ChIA-PET datasets permitted the independent rediscovery of known trends in chromatin loops including enrichment of CTCF, RAD21, SMC3 and ZNF143 at the anchor regions of interactions and strong bias for convergent CTCF motifs. Mango is open source and distributed through github at https://github.com/dphansti/mango. mpsnyder@standford.edu Supplementary data are available at Bioinformatics online.

A Novel Enhancer Region 1.7Mb Downstream of the C-Myc Gene Drives Its Expression in Hematopoietic Stem and Progenitor Cells

The c-MYC transcription factor is a central regulator of cellular proliferation, growth, metabolism and differentiation in many cell types including stem cells (1). Although it is known that c-Myc expression is tightly controlled and can drive transformation if de-regulated, the mechanisms of its transcriptional regulation remain elusive. Besides its promoter, which is not sufficient to account for Myc endogenous expression, only a few cis-regulatory elements have been defined. c-Myc is located within a 4 Mb-long gene-poor region, which coincides with a large topologically associating domain (TAD) (2). At the distal end of this TAD, 1.7 Mb downstream of the mouse c-Myc gene, we identified a cluster of enhancer-associated chromatin marks which were present only in hematopoietic tissues. A LacZ reporter gene inserted next to this cluster showed specific expression in hematopoietic stem cells (HSCs) and progenitor cells. Mice homozygous for a deletion of this enhancer region showed decreased myeloid and B cells, while HSCs, multipotent progenitors and megakaryocytes accumulated in the bone marrow. This phenotype closely mimicked the phenotype of mice in which the c-Myc gene was conditionally deleted using Mx1-Cre (3). Importantly, gene expression analysis showed that the deletion of this enhancer region led to a dramatic reduction of Myc expression in HSCs, multipotent progenitors and most mature cell types. Furthermore, compound heterozygous mice carrying one enhancer deletion allele and one c-Myc null allele demonstrated very similar hematopoietic defects to that of homozygous c-Myc null mice, showing allelism between c-Myc and the enhancer region. Altogether, these data provide genetic evidence that this enhancer region directly controls, in cis, c-Myc expression in HSC/progenitor cells. As the region is composed of multiple modules, we examined the relative enrichment of the enhancer-associated H3K27ac mark by ChIP for the different modules in different hematopoietic lineages. This analysis suggested that selective enhancer elements contribute differently to c-Myc expression in either granulocytes or HSC/progenitors. Interestingly, this highly conserved enhancer region is focally amplified in a number of AML patients, suggesting that it may be a critical component driving the increased Myc expression found in leukemias. In summary, we identified a distant hematopoietic-specific enhancer region for c-Myc and provide data which supports its critical function as a key regulatory region in normal hematopoiesis and highlights a potential role in leukemic transformation.(1) Laurenti et al. (2008). Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. Cell Stem Cell 3(6): 611-624.(2) Dixon et al. (2012). Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485(7398): 376-380.(3) Wilson et al. (2004). c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev 18(22): 2747-2763. DisclosuresNo relevant conflicts of interest to declare.

A Bayesian mixture model for chromatin interaction data.

Chromatin interactions mediated by a particular protein are of interest for studying gene regulation, especially the regulation of genes that are associated with, or known to be causative of, a disease. A recent molecular technique, Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), that uses chromatin immunoprecipitation (ChIP) and high throughput paired-end sequencing, is able to detect such chromatin interactions genomewide. However, ChIA-PET may generate noise (i.e., pairings of DNA fragments by random chance) in addition to true signal (i.e., pairings of DNA fragments by interactions). In this paper, we propose MC_DIST based on a mixture modeling framework to identify true chromatin interactions from ChIA-PET count data (counts of DNA fragment pairs). The model is cast into a Bayesian framework to take into account the dependency among the data and the available information on protein binding sites and gene promoters to reduce false positives. A simulation study showed that MC_DIST outperforms the previously proposed hypergeometric model in terms of both power and type I error rate. A real data study showed that MC_DIST may identify potential chromatin interactions between protein binding sites and gene promoters that may be missed by the hypergeometric model. An R package implementing the MC_DIST model is available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM.

Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing technology and application.

BackgroundLong-range chromatin interactions play an important role in transcription regulation. Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET) is an emerging technology that has unique advantages in chromatin interaction analysis, and thus provides insight into the study of transcription regulation.ResultsThis article introduces the experimental protocol and data analysis process of ChIA-PET, as well as discusses some applications using this technology. It also unveils the direction of future studies based on this technology.ConclusionsOverall we show that ChIA-PET is the cornerstone to explore the three-dimensional (3D) chromatin structure, and certainly will lead the forthcoming wave of 3D genomics studies.