Stefan Schoenfelder

Spatiotemporal gene expression programmes are orchestrated by transcriptional enhancers, which are key regulatory DNA elements that engage in physical contacts with their target-gene promoters, often bridging considerable genomic distances. Recent progress in genomics, genome editing and microscopy methodologies have enabled the genome-wide mapping of enhancer–promoter contacts and their functional dissection. In this Review, we discuss novel concepts on how enhancer–promoter interactions are… Show more

Spatiotemporal gene expression programmes are orchestrated by transcriptional enhancers, which are key regulatory DNA elements that engage in physical contacts with their target-gene promoters, often bridging considerable genomic distances. Recent progress in genomics, genome editing and microscopy methodologies have enabled the genome-wide mapping of enhancer–promoter contacts and their functional dissection. In this Review, we discuss novel concepts on how enhancer–promoter interactions are established and maintained, how the 3D architecture of mammalian genomes both facilitates and constrains enhancer–promoter contacts, and the role they play in gene expression control during normal development and disease. Show less

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells

The establishment of the embryonic and trophoblast lineages is a developmental decision underpinned by dramatic differences in the epigenetic landscape of the two compartments. However, it remains unknown how epigenetic information and transcription factor networks map to the 3D arrangement of the genome, which in turn may mediate transcriptional divergence between the two cell lineages. Here, we perform promoter capture Hi-C experiments in mouse trophoblast (TSC) and embryonic (ESC) stem cells… Show more

The establishment of the embryonic and trophoblast lineages is a developmental decision underpinned by dramatic differences in the epigenetic landscape of the two compartments. However, it remains unknown how epigenetic information and transcription factor networks map to the 3D arrangement of the genome, which in turn may mediate transcriptional divergence between the two cell lineages. Here, we perform promoter capture Hi-C experiments in mouse trophoblast (TSC) and embryonic (ESC) stem cells to understand how chromatin conformation relates to cell-specific transcriptional programmes. We find that key TSC genes that are kept repressed in ESCs exhibit interactions between H3K27me3-marked regions in ESCs that depend on Polycomb repressive complex 1. Interactions that are prominent in TSCs are enriched for enhancer–gene contacts involving key TSC transcription factors, as well as TET1, which helps to maintain the expression of TSC-relevant genes. Our work shows that the first developmental cell fate decision results in distinct chromatin conformation patterns establishing lineage-specific contexts involving both repressive and active interactions. Show less

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported. Here, we used… Show more

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported. Here, we used promoter-capture Hi-C to identify promoters that interact with SEs in mouse embryonic stem cells (ESCs). We found that SEs form complex, spatial networks in which individual SEs contact multiple promoters, and a rewiring of promoter-SE interactions occurs between pluripotent states. We also show that long-range promoter-SE interactions are more prevalent in ESCs than in epiblast stem cells (EpiSCs) or Nanog-deficient ESCs. We conclude that SEs form cell-type-specific interaction networks that are partly dependent on core transcription factors, thereby providing insights into the gene regulatory organization of pluripotent cells. Show less

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relationship between cis-regulatory activity and… Show more

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relationship between cis-regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 min of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO. Show less

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF… Show more

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome‐wide function in mediating long‐range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL. Show less

Chromosomal rearrangements occur constitutionally in the general population and somatically in the majority of cancers. Detection of balanced rearrangements, such as reciprocal translocations and inversions, is troublesome, which is particularly detrimental in oncology where rearrangements play diagnostic and prognostic roles. Here we describe the use of Hi-C as a tool for detection of both balanced and unbalanced chromosomal rearrangements in primary human tumour samples, with the potential to… Show more

Chromosomal rearrangements occur constitutionally in the general population and somatically in the majority of cancers. Detection of balanced rearrangements, such as reciprocal translocations and inversions, is troublesome, which is particularly detrimental in oncology where rearrangements play diagnostic and prognostic roles. Here we describe the use of Hi-C as a tool for detection of both balanced and unbalanced chromosomal rearrangements in primary human tumour samples, with the potential to define chromosome breakpoints to bp resolution. In addition, we show copy number profiles can also be obtained from the same data, all at a significantly lower cost than standard sequencing approaches. Show less

Hi-C is one of the main methods for investigating spatial co-localisation of DNA in the nucleus. However, the raw sequencing data obtained from Hi-C experiments suffer from large biases and spurious contacts, making it difficult to identify true interactions. Existing methods use complex models to account for biases and do not provide a significance threshold for detecting interactions. Here we introduce a simple binomial probabilistic model that resolves complex biases and distinguishes… Show more

Hi-C is one of the main methods for investigating spatial co-localisation of DNA in the nucleus. However, the raw sequencing data obtained from Hi-C experiments suffer from large biases and spurious contacts, making it difficult to identify true interactions. Existing methods use complex models to account for biases and do not provide a significance threshold for detecting interactions. Here we introduce a simple binomial probabilistic model that resolves complex biases and distinguishes between true and false interactions. The model corrects biases of known and unknown origin and yields a p-value for each interaction, providing a reliable threshold based on significance. We demonstrate this experimentally by testing the method against a random ligation dataset. Our method outperforms previous methods and provides a statistical framework for further data analysis, such as comparisons of Hi-C interactions between different conditions. GOTHiC is available as a BioConductor package (http://www.bioconductor.org/packages/release/bioc/html/GOTHiC.html). Show less

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… Show more

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.
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The identification of causal genes from genome-wide association studies (GWAS) is the next important step for the translation of genetic findings into biologically meaningful mechanisms of disease and potential therapeutic targets. Using novel chromatin interaction detection techniques and allele specific assays in T and B cell lines, we provide compelling evidence that redefines causal genes at the 6q23 locus, one of the most important loci that confers autoimmunity risk. Although the function… Show more

The identification of causal genes from genome-wide association studies (GWAS) is the next important step for the translation of genetic findings into biologically meaningful mechanisms of disease and potential therapeutic targets. Using novel chromatin interaction detection techniques and allele specific assays in T and B cell lines, we provide compelling evidence that redefines causal genes at the 6q23 locus, one of the most important loci that confers autoimmunity risk. Although the function of disease-associated non-coding single nucleotide polymorphisms (SNPs) at 6q23 is unknown, the association is generally assigned to TNFAIP3, the closest gene. However, the DNA fragment containing the associated SNPs interacts through chromatin looping not only with TNFAIP3, but also with IL20RA, located 680 kb upstream. The risk allele of the most likely causal SNP, rs6927172, is correlated with both a higher frequency of interactions and increased expression of IL20RA, along with a stronger binding of both the NFκB transcription factor and chromatin marks characteristic of active enhancers in T-cells. Our results highlight the importance of gene assignment for translating GWAS findings into biologically meaningful mechanisms of disease and potential therapeutic targets; indeed, monoclonal antibody therapy targeting IL-20 is effective in the treatment of rheumatoid arthritis and psoriasis, both with strong GWAS associations to this region. Show less

Capture Hi-C (CHi-C) is a method for profiling chromosomal interactions involving targeted regions of interest, such as gene promoters, globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments. We implement these procedures in CHiCAGO (… Show more

Capture Hi-C (CHi-C) is a method for profiling chromosomal interactions involving targeted regions of interest, such as gene promoters, globally and at high resolution. Signal detection in CHi-C data involves a number of statistical challenges that are not observed when using other Hi-C-like techniques. We present a background model and algorithms for normalisation and multiple testing that are specifically adapted to CHi-C experiments. We implement these procedures in CHiCAGO ( http://regulatorygenomicsgroup.org/chicago ), an open-source package for robust interaction detection in CHi-C. We validate CHiCAGO by showing that promoter-interacting regions detected with this method are enriched for regulatory features and disease-associated SNPs. Show less

Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic… Show more

Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor–dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology. Show less

Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare… Show more

Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation. Show less

Genome-wide association studies have been tremendously successful in identifying genetic variants associated with complex diseases. The majority of association signals are intergenic and evidence is accumulating that a high proportion of signals lie in enhancer regions. We use Capture Hi-C to investigate, for the first time, the interactions between associated variants for four autoimmune diseases and their functional targets in B- and T-cell lines. Here we report numerous looping interactions… Show more

Genome-wide association studies have been tremendously successful in identifying genetic variants associated with complex diseases. The majority of association signals are intergenic and evidence is accumulating that a high proportion of signals lie in enhancer regions. We use Capture Hi-C to investigate, for the first time, the interactions between associated variants for four autoimmune diseases and their functional targets in B- and T-cell lines. Here we report numerous looping interactions and provide evidence that only a minority of interactions are common to both B- and T-cell lines, suggesting interactions may be highly cell-type specific; some disease-associated SNPs do not interact with the nearest gene but with more compelling candidate genes (for example, FOXO1, AZI2) often situated several megabases away; and finally, regions associated with different autoimmune diseases interact with each other and the same promoter suggesting common autoimmune gene targets (for example, PTPRC, DEXI and ZFP36L1). Show less

HiCUP is a pipeline for processing sequence data generated by Hi-C and Capture Hi-C (CHi-C) experiments, which are techniques used to investigate three-dimensional genomic organisation. The pipeline maps data to a specified reference genome and removes artefacts that would otherwise hinder subsequent analysis. HiCUP also produces an easy-to-interpret yet detailed quality control (QC) report that assists in refining experimental protocols for future studies. The software is freely available and… Show more

HiCUP is a pipeline for processing sequence data generated by Hi-C and Capture Hi-C (CHi-C) experiments, which are techniques used to investigate three-dimensional genomic organisation. The pipeline maps data to a specified reference genome and removes artefacts that would otherwise hinder subsequent analysis. HiCUP also produces an easy-to-interpret yet detailed quality control (QC) report that assists in refining experimental protocols for future studies. The software is freely available and has already been used for processing Hi-C and CHi-C data in several recently published peer-reviewed studies. Show less

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… Show more

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. Show less

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… Show more

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. Show less

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… Show more

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. Show less

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer–promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C… Show more

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer–promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci. Show less

Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular… Show more

Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation. Show less

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual… Show more

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. Show less

In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin… Show more

In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs. Show less

Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by… Show more

Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements (“bait fragments”) within the captured regions and “targets” that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant. Show less

Transcription in the eukaryotic nucleus has long been thought of as conforming to a model in which RNA polymerase complexes are recruited to and track along isolated templates. However, a more dynamic role for chromatin in transcriptional regulation is materializing: enhancer elements interact with promoters forming loops that often bridge considerable distances and genomic loci, even located on different chromosomes, undergo chromosomal associations. These associations amass to form an… Show more

Transcription in the eukaryotic nucleus has long been thought of as conforming to a model in which RNA polymerase complexes are recruited to and track along isolated templates. However, a more dynamic role for chromatin in transcriptional regulation is materializing: enhancer elements interact with promoters forming loops that often bridge considerable distances and genomic loci, even located on different chromosomes, undergo chromosomal associations. These associations amass to form an extensive ‘transcriptional interactome’, enacted at functional subnuclear compartments, to which genes dynamically relocate. The emerging view is that long-range chromosomal associations between genomic regions, and their repositioning in the three-dimensional space of the nucleus, are key contributors to the regulation of gene expression. Show less

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate… Show more

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control. Show less

The imprinting control region (ICR) upstream of H19 is the key regulatory element conferring monoallelic expression on H19 and Igf2 (insulin‐like growth factor 2). Epigenetic marks in the ICR regulate its interaction with the chromatin protein CCCTC‐binding factor and with other control factors to coordinate gene silencing in the imprinting cluster. Here, we show that the H19 ICR is biallelically transcribed, producing both sense and antisense RNAs. We analyse the function of the non‐coding… Show more

The imprinting control region (ICR) upstream of H19 is the key regulatory element conferring monoallelic expression on H19 and Igf2 (insulin‐like growth factor 2). Epigenetic marks in the ICR regulate its interaction with the chromatin protein CCCTC‐binding factor and with other control factors to coordinate gene silencing in the imprinting cluster. Here, we show that the H19 ICR is biallelically transcribed, producing both sense and antisense RNAs. We analyse the function of the non‐coding transcripts in a Drosophila transgenic system in which the H19 upstream region silences the expression of a reporter gene. We show that knockdown of H19 ICR non‐coding RNA (ncRNA) by RNA interference leads to the loss of reporter gene silencing. Our results are, to the best of our knowledge, the first to show that ncRNAs in the H19 ICR are functionally significant, and also indicate that they have a role in regulating gene expression and perhaps epigenetic marks at the H19/Igf2 locus. Show less

Two alleles of the Drosophila melanogaster Rfc4(DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating… Show more

Two alleles of the Drosophila melanogaster Rfc4(DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these twoDmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes. Show less