Cohesin-dependent regulation of gene expression during differentiation is lost in cohesin-mutated myeloid malignancies.

Cohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies.

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Capturing genomic relationships that matter.

There is a strong interrelationship within the cell nucleus between form and function of the genome. This connection is exhibited across multiple hierarchies, ranging from grand-scale positioning of chromosomes and their intersection with specific nuclear functional activities, the segregation of chromosome structure into distinct domains and long-range regulatory contacts that drive spatial and temporal expression patterns of genes. Fifteen years ago, the development of the chromosome conformation capture method placed the nature of specific, long-range regulatory interactions under scrutiny. However, its development and integration with next-generation sequencing technologies has greatly expanded the breadth and scope of what is detected. The sheer scale of data offered by these important advances has come with new and challenging bottlenecks that are both experimental and bioinformatical. Here, we discuss the recent and prospective development and implementation of new methodologies and analytical tools that are allowing an in-depth, yet focussed characterisation of genomic contacts that are associated with functional activities in the nucleus.

Long-range chromatin regulatory interactions in vivo.

Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

Active genes dynamically colocalize to shared sites of ongoing transcription.

The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites.

Multi-omics and machine learning reveal context-specific gene regulatory activities of PML::RARA in acute promyelocytic leukemia.

The PML::RARA fusion protein is the hallmark driver of Acute Promyelocytic Leukemia (APL) and disrupts retinoic acid signaling, leading to wide-scale gene expression changes and uncontrolled proliferation of myeloid precursor cells. While known to be recruited to binding sites across the genome, its impact on gene regulation and expression is under-explored. Using integrated multi-omics datasets, we characterize the influence of PML::RARA binding on gene expression and regulation in an inducible PML::RARA cell line model and APL patient ex vivo samples. We find that genes whose regulatory elements recruit PML::RARA are not uniformly transcriptionally repressed, as commonly suggested, but also may be upregulated or remain unchanged. We develop a computational machine learning implementation called Regulatory Element Behavior Extraction Learning to deconvolute the complex, local transcription factor binding site environment at PML::RARA bound positions to reveal distinct signatures that modulate how PML::RARA directs the transcriptional response.

Comparison of Capture Hi-C Analytical Pipelines.

It is now evident that DNA forms an organized nuclear architecture, which is essential to maintain the structural and functional integrity of the genome. Chromatin organization can be systematically studied due to the recent boom in chromosome conformation capture technologies (e.g., 3C and its successors 4C, 5C and Hi-C), which is accompanied by the development of computational pipelines to identify biologically meaningful chromatin contacts in such data. However, not all tools are applicable to all experimental designs and all structural features. Capture Hi-C (CHi-C) is a method that uses an intermediate hybridization step to target and select predefined regions of interest in a Hi-C library, thereby increasing effective sequencing depth for those regions. It allows researchers to investigate fine chromatin structures at high resolution, for instance promoter-enhancer loops, but it introduces additional biases with the capture step, and therefore requires specialized pipelines. Here, we compare multiple analytical pipelines for CHi-C data analysis. We consider the effect of retaining multi-mapping reads and compare the efficiency of different statistical approaches in both identifying reproducible interactions and determining biologically significant interactions. At restriction fragment level resolution, the number of multi-mapping reads that could be rescued was negligible. The number of identified interactions varied widely, depending on the analytical method, indicating large differences in type I and type II error rates. The optimal pipeline depends on the project-specific tolerance level of false positive and false negative chromatin contacts.

Mutational synergy during leukemia induction remodels chromatin accessibility, histone modifications and three-dimensional DNA topology to alter gene expression.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.

Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.

Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.

Raising the curtains on interchromosomal interactions.

The chromosome conformation capture technique is used to monitor intra- and intermolecular chromosomal associations. By introducing an adaptation of this technique, Ling and colleagues have identified an unexpected coassociation between two loci on separate chromosomes in mouse nuclei, the imprinted Igf2-H19 locus of chromosome 7 and the Wsb1-Nf1 locus of chromosome 11. Strikingly, this interaction is CCCTC-binding factor (CTCF)-dependent and strictly allele specific. These findings extend our appreciation for genome organization and its influence on gene expression and imprinting.

RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction.

Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1-ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1-ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors.

An intergenic non-coding RNA promoter required for histone modifications in the human ß-globin chromatin domain.

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human ß-globin genes. Mutational analyses in ß-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and ß-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of ß-like globin gene transcription during development.

Where shall we meet? A role for genome organisation and nuclear sub-compartments in mediating interchromosomal interactions.

A recent spate of examples of specific interactions between loci on separate chromosomes in mammalian nuclei has illuminated another layer of complexity in gene regulation. As the specifics of the cross-talk between interacting loci are worked out, it is also important to consider exactly how, when and where loci can ever reliably find each other within such an intricate environment. Answers may lie in how the genome is organised in relation to itself and to specialised nuclear sub-compartments. Here, we discuss how such specialised nuclear bodies may have the potential to specifically sequester loci and provide a context where interchromosomal communications can occur.

Molecular determinants of NOTCH4 transcription in vascular endothelium.

The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.

Promoter capture Hi-C-based identification of recurrent noncoding mutations in colorectal cancer.

Efforts are being directed to systematically analyze the non-coding regions of the genome for cancer-driving mutations1-6. cis-regulatory elements (CREs) represent a highly enriched subset of the non-coding regions of the genome in which to search for such mutations. Here we use high-throughput chromosome conformation capture techniques (Hi-C) for 19,023 promoter fragments to catalog the regulatory landscape of colorectal cancer in cell lines, mapping CREs and integrating these with whole-genome sequence and expression data from The Cancer Genome Atlas7,8. We identify a recurrently mutated CRE interacting with the ETV1 promoter affecting gene expression. ETV1 expression influences cell viability and is associated with patient survival. We further refine our understanding of the regulatory effects of copy-number variations, showing that RASL11A is targeted by a previously identified enhancer amplification1. This study reveals new insights into the complex genetic alterations driving tumor development, providing a paradigm for employing chromosome conformation capture to decipher non-coding CREs relevant to cancer biology.

Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells.

Long-range cis-regulatory elements such as enhancers coordinate cell-specific transcriptional programmes by engaging in DNA looping interactions with target promoters. Deciphering the interplay between the promoter connectivity and activity of cis-regulatory elements during lineage commitment is crucial for understanding developmental transcriptional control. Here, we use Promoter Capture Hi-C to generate a high-resolution atlas of chromosomal interactions involving ~22,000 gene promoters in human pluripotent and lineage-committed cells, identifying putative target genes for known and predicted enhancer elements. We reveal extensive dynamics of cis-regulatory contacts upon lineage commitment, including the acquisition and loss of promoter interactions. This spatial rewiring occurs preferentially with predicted changes in the activity of cis-regulatory elements and is associated with changes in target gene expression. Our results provide a global and integrated view of promoter interactome dynamics during lineage commitment of human pluripotent cells.

Retrovirus silencer blocking by the cHS4 insulator is CTCF independent.

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.

MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs.

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.

Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C.

Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type-restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.