Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

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title: Systematic analysis of noncoding somatic mutations and gene expression alterations across 14 tumor types
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title: Role of non-coding sequence variants in cancer
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title: Developmental Fate and Cellular Maturity Encoded in Human Regulatory DNA Landscapes
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title: Enhancer deregulation in cancer and other diseases
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title: Control of Cell Identity Genes Occurs in Insulated Neighborhoods in Mammalian Chromosomes
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title: CTCF: the protein, the binding partners, the binding sites and their chromatin loops
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title: Cohesin relocation from sites of chromosomal loading to places of convergent transcription
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title: CTCF: an architectural protein bridging genome topology and function
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title: Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions
abstract: Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

title: Functional analysis of CTCF during mammalian limb development.
abstract: CCCTC-binding factor (CTCF) is a nuclear zinc-finger protein that displays insulating activity in a variety of biological assays. For example, CTCF-binding sites have been suggested to isolate Hox gene clusters from neighboring transcriptional interference. We investigated this issue during limb development, where Hoxd genes must remain isolated from long-range effects to allow essential regulation within independent sub-groups. We used conditional Ctcf inactivation in incipient forelimbs and show that the overall pattern of Hoxd gene expression remains unchanged. Transcriptome analysis using tiling arrays covering chromosomes 2 and X confirmed the weak effect of CTCF depletion on global gene regulation. However, Ctcf deletion caused massive apoptosis, leading to a nearly complete loss of limb structure at a later stage. We conclude that, at least in this physiological context, rather than being an insulator, CTCF is required for cell survival via the direct transcriptional regulation of target genes critical for cellular homeostasis.

title: CTCF Binding Polarity Determines Chromatin Looping.
abstract: CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.

title: Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes.
abstract: Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (-45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the -45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the -45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs.

title: CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation
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title: CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function
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title: The DNA-binding factor Ctcf critically controls gene expression in macrophages
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title: CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus.
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title: Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes
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title: Activation of proto-oncogenes by disruption of chromosome neighborhoods
abstract: The spread of bad neighborhoods Our genomes have complex three-dimensional (3D) arrangements that partition and regulate gene expression. Cancer cells frequently have their genomes grossly rearranged, disturbing this intricate 3D organization. Hnisz et al. show that the disruption of these 3D neighborhoods can bring oncogenes under the control of regulatory elements normally kept separate from them (see the Perspective by Wala and Beroukim). These novel juxtapositions can result in the inappropriate activation of oncogenes. Science, this issue p. 1454; see also p. 1398 Disrupting the boundaries between three-dimensional neighborhoods in the genome can activate cancer-promoting genes.  Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.

title: Formation of new chromatin domains determines pathogenicity of genomic duplications
abstract: Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

title: Epigenetic Modifications Unlock the Milk Protein Gene Loci during Mouse Mammary Gland Development and Differentiation
abstract: Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation.  Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization.  A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland.

title: Terminal differentiation of goat mammary tissue during pregnancy requires the expression of genes involved in immune functions.
abstract: Terminal differentiation of mammary tissue into a functional epithelium that synthesizes and secretes milk occurs during pregnancy. The molecular mechanisms underlying this complex process are poorly understood, especially in ruminants. To obtain an overview of the ruminant mammary gland's final differentiation process, we conducted time-course gene expression analysis of five physiological stages: four during pregnancy (P46, P70, P90, and P110) and one after 40 days of lactation (L40). An appropriate loop experimental design was used to follow gene expression profiles. Using three nulliparous (pregnancy) or primiparous (lactation) goats per stage, we performed a comparison starting from nine dye-swaps and using a 22K bovine oligoarray. Statistical analysis revealed that the expression of 1,696 genes varied significantly at least once in the study. These genes fell into 19 clusters based on their expression profiles. Identification of biological functions with Ingenuity Pathway Analysis software revealed several similarities, in keeping with physiological stages described in mice. As in mice, expression of milk protein genes began at midpregnancy, and genes regulating lipid biosynthesis were induced at the onset of lactation. During the first half of pregnancy, the molecular signature of goat mammary tissue was characterized by the expression of genes associated with tissue remodeling and differentiation, while the second half was mainly characterized by the presence of messengers encoding genes involved in cell proliferation. A large number of immune-related genes were also induced, supporting recent speculation that mammary tissue has an original immune function, and the recruitment of migrating hematopoietic cells possibly involved in the branching morphogenesis of the mammary gland. These data hint that the induction of differentiation occurs early in pregnancy, very likely before P46. This period is therefore crucial for obtaining a healthy and productive mammary gland.

title: Mammary-Specific Gene Activation Is Defined by Progressive Recruitment of STAT5 during Pregnancy and the Establishment of H3K4me3 Marks
abstract: Differentiation of mammary secretory epithelium during pregnancy is characterized by sequential activation of genes over several orders of magnitude. Although the transcription factor STAT5 is key to alveolar development, it is not clear to what extent it controls temporal activation of genetic programs in secretory epithelium. To uncover molecular mechanisms effecting progressive differentiation, we explored genome-wide STAT5 binding and H3K4me3 (i.e., trimethylated histone H3 at K4) marks in mammary tissues at early and midpregnancy and at parturition. STAT5 binding to genes induced during pregnancy was low in immature mammary tissue but increased with epithelial differentiation. Increased STAT5 binding was associated with the establishment of H3K4me3 marks and transcriptional activation. STAT5 binding preceded the formation of H3K4me3 marks in some mammary-specific genes. De novo STAT5 binding was also found at distal sites, indicating enhancers. Furthermore, we established an exhaustive mammary transcriptome. Through integration of RNA-seq and STAT5 and H3K4me4 ChIP-seq data, we discovered novel mammary-specific alternative promoters and genes, including noncoding RNAs. Our findings suggest that STAT5 is an early step in establishing transcription complexes on genes specifically expressed in mammary epithelium. This is the first study in an organ that links progressive chromatin occupancy of STAT5 to the acquisition of H3K4me3 marks and transcription during hormone-induced differentiation.

title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation
abstract: Cytokines utilize the transcription factor STAT5 to control cell-specific genes at a larger scale than universal genes, with a mechanistic explanation yet to be supplied. Genome-wide studies have identified putative STAT5-based mammary-specific and universal enhancers, an opportunity to investigate mechanisms underlying their differential response to cytokines. We have now interrogated the integrity and function of both categories of regulatory elements using biological and genetic approaches. During lactation, STAT5 occupies mammary-specific and universal cytokine-responsive elements. Following lactation, prolactin levels decline and mammary-specific STAT5-dependent enhancers are decommissioned within 24 h, while universal regulatory complexes remain intact. These differential sensitivities are linked to STAT5 concentrations and the mammary-specific Stat5 autoregulatory enhancer. In its absence, mammary-specific enhancers, but not universal elements, fail to be fully established. Upon termination of lactation STAT5 binding to a subset of mammary enhancers is substituted by STAT3. No STAT3 binding was observed at the most sensitive STAT5 enhancers suggesting that upon hormone withdrawal their chromatin becomes inaccessible. Lastly, we demonstrate that the mammary-enriched transcription factors GR, ELF5 and NFIB associate with STAT5 at sites lacking bona fide binding motifs. This study provides, for the first time, molecular insight into the differential sensitivities of mammary-specific and universal cytokine-sensing enhancers.

title: Analysis of long-range chromatin interactions using Chromosome Conformation Capture.
abstract: Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.

title: Quantitative analysis of chromosome conformation capture assays (3C-qPCR)
abstract: Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7–9 days.

title: Multiplexed chromosome conformation capture sequencing for rapid genome-scale high-resolution detection of long-range chromatin interactions
abstract: Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1–8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

title: Detecting long-range chromatin interactions using the chromosome conformation capture sequencing (4C-seq) method.
abstract: Eukaryotic transcription is tightly regulated by transcriptional regulatory elements, even though these elements may be located far away from their target genes. It is now widely recognized that these regulatory elements can be brought in close proximity through the formation of chromatin loops, and that these loops are crucial for transcriptional regulation of their target genes. The chromosome conformation capture (3C) technique presents a snapshot of long-range interactions, by fixing physically interacting elements with formaldehyde, digestion of the DNA, and ligation to obtain a library of unique ligation products. Recently, several large-scale modifications to the 3C technique have been presented. Here, we describe chromosome conformation capture sequencing (4C-seq), a high-throughput version of the 3C technique that combines the 3C-on-chip (4C) protocol with next-generation Illumina sequencing. The method is presented for use in mammalian cell lines, but can be adapted to use in mammalian tissues and any other eukaryotic genome.

title: 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments
abstract: 4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or “bait”) that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.

title: Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression
abstract: The mechanism by which prolactin, a peptide hormone, regulates casein gene expression has been studied in mammary gland organ culture. After prolactin addition, a 2-4 fold increase in the rate of casein mRNA transcription was observed within 1 hr and maintained for at least 24 hr. This increased rate of transcription is not sufficient to account for the mass accumulation of casein mRNA. The half-life of casein mRNA is also increased 17-25 fold in the presence of prolactin. This change in casein mRNA half-life, coupled with a 2-4 fold increase in the rate of transcription, can account for the normal accumulation of casein mRNA observed after prolactin addition. This hormone-induced change in casein mRNA half-life appeared to be selective, since prolactin was found to exert only a slight effect (1-4 fold) on the half-life of poly(A) RNA determined under identical pulse-chase conditions. The hormonal regulation of casein gene expression thus does not app-ar to be an "all or none" process occurring only at the transcriptional or post-transcriptional levels, but rather may involve a coordinated response at several levels to permit the efficient expression of specialized differentiated functions.

title: Expression pattern, subcellular localization, and functional implications of ODAM in ameloblasts, odontoblasts, osteoblasts, and various cancer cells.
abstract: During tooth development and tumorigenesis, the odontogenic ameloblast-associated protein (ODAM) is involved in cellular differentiation and matrix protein production. However, the precise function of ODAM remains largely unknown. To suggest new functional roles of ODAM, we investigated the cellular expression and subcellular localization of ODAM in tooth and cancer cells. ODAM was expressed in ameloblasts, odontoblasts, and osteoblasts in vivo and in vitro. Furthermore, ODAM was localized in both the nucleus and cytoplasm of MMP-20 expressing ameloblasts and odontoblasts, but only in the cytoplasm of non-MMP-20 expressing osteoblasts. The extracellular secretion of ODAM was not observed in odontoblasts and osteoblasts, but was seen in ameloblasts. In addition, ODAM was discovered in the nucleus, cytoplasm, and extracellular matrix of various cancer cells. These results suggest that the expression pattern and subcellular localization of ODAM is highly variable and dependent on cell types and their differentiation states, and that functional correlations exist between ODAM and MMP-20. This study provides the first evidence for ODAM in multiple cellular compartments of differentiating odontogenic and cancer cell lines with important functional implications.

title: CHiCAGO: Robust Detection of DNA Looping Interactions in Capture Hi-C data
abstract: Capture Hi-C (CHi-C) is a state-of-the art 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, in which many spatially dispersed regions are captured, such as in Promoter CHi-C. We implement these procedures in 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.

title: Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions
abstract: The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term ‘topological domains’, as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.

title: Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus
abstract: Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.

title: Dynamic Control of Enhancer Repertoires Drives Lineage and Stage-Specific Transcription during Hematopoiesis.
abstract: Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.

title: CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription
abstract: 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.

title: CTCF Binding Elements Mediate Control of V(D)J Recombination
abstract: Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the VH and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VH gene segments and promoting rearrangement of distal VH segments. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination by suppressing VH joining to D segments not joined to JH segments, and VH to DJH joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal VH-to-DJH recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.

title: Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development
abstract: Zhao and collaborators use linear amplification–mediated high-throughput genome-wide translocation sequencing to examine TCRδ VDJ recombination at an unprecedented resolution.

title: Editing DNA Methylation in the Mammalian Genome
abstract: Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

title: CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells
abstract: HOX cluster genes are activated sequentially in their positional order along the chromosome during vertebrate development. This phenomenon, known as temporal colinearity, depends on transcriptional silencing of 5′ HOX genes. Chromatin looping was recently identified as a conserved feature of silent HOX clusters, with CCCTC-binding factor (CTCF) binding sites located at the loop bases. However, the potential contribution of CTCF to HOX cluster silencing and the underlying mechanism have not been established. Here, we demonstrate that the HOXA locus is organized by CTCF into chromatin loops and that CTCF depletion causes significantly enhanced activation of HOXA3 to -A7, -A9 to -A11, and -A13 in response to retinoic acid, with the highest effect observed for HOXA9. Our subsequent analyses revealed that CTCF facilitates the stabilization of Polycomb repressive complex 2 (PRC2) and trimethylated lysine 27 of histone H3 (H3K27me3) at the human HOXA locus. Our results reveal that CTCF functions as a controller of HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure.

title: A Variably Occupied CTCF Binding Site in the Ultrabithorax Gene in the Drosophila Bithorax Complex
abstract: Although the majority of genomic binding sites for the insulator protein CCCTC-binding factor (CTCF) are constitutively occupied, a subset show variable occupancy. Such variable sites provide an opportunity to assess context-specific CTCF functions in gene regulation. Here, we have identified a variably occupied CTCF site in the Drosophila Ultrabithorax (Ubx) gene. This site is occupied in tissues where Ubx is active (third thoracic leg imaginal disc) but is not bound in tissues where the Ubx gene is repressed (first thoracic leg imaginal disc). Using chromatin conformation capture, we show that this site preferentially interacts with the Ubx promoter region in the active state. The site lies close to Ubx enhancer elements and is also close to the locations of several gypsy transposon insertions that disrupt Ubx expression, leading to the bx mutant phenotype. gypsy insertions carry the Su(Hw)-dependent gypsy insulator and were found to affect both CTCF binding at the variable site and the chromatin topology. This suggests that insertion of the gypsy insulator in this region interferes with CTCF function and supports a model for the normal function of the variable CTCF site as a chromatin loop facilitator, promoting interaction between Ubx enhancers and the Ubx transcription start site.

title: CTCF-mediated topological boundaries during development foster appropriate gene regulation.
abstract: The genome is organized into repeating topologically associated domains (TADs), each of which is spatially isolated from its neighbor by poorly understood boundary elements thought to be conserved across cell types. Here, we show that deletion of CTCF (CCCTC-binding factor)-binding sites at TAD and sub-TAD topological boundaries that form within the HoxA and HoxC clusters during differentiation not only disturbs local chromatin domain organization and regulatory interactions but also results in homeotic transformations typical of Hox gene misregulation. Moreover, our data suggest that CTCF-dependent boundary function can be modulated by competing forces, such as the self-assembly of polycomb domains within the nucleus. Therefore, CTCF boundaries are not merely static structural components of the genome but instead are locally dynamic regulatory structures that control gene expression during development.

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

title: The Epigenetic Landscape of Mammary Gland Development and Functional Differentiation
abstract: Most of the development and functional differentiation in the mammary gland occur after birth. Epigenetics is defined as the stable alterations in gene expression potential that arise during development and proliferation. Epigenetic changes are mediated at the biochemical level by the chromatin conformation initiated by DNA methylation, histone variants, post-translational modifications of histones, non-histone chromatin proteins, and non-coding RNAs. Epigenetics plays a key role in development. However, very little is known about its role in the developing mammary gland or how it might integrate the many signalling pathways involved in mammary gland development and function that have been discovered during the past few decades. An inverse relationship between marks of closed (DNA methylation) or open chromatin (DnaseI hypersensitivity, certain histone modifications) and milk protein gene expression has been documented. Recent studies have shown that during development and functional differentiation, both global and local chromatin changes occur. Locally, chromatin at distal regulatory elements and promoters of milk protein genes gains a more open conformation. Furthermore, changes occur both in looping between regulatory elements and attachment to nuclear matrix. These changes are induced by developmental signals and environmental conditions. Additionally, distinct epigenetic patterns have been identified in mammary gland stem and progenitor cell sub-populations. Together, these findings suggest that epigenetics plays a role in mammary development and function. With the new tools for epigenomics developed in recent years, we now can begin to establish a framework for the role of epigenetics in mammary gland development and disease.

title: Allele-Specific Binding of CTCF to the Multipartite Imprinting Control Region KvDMR1
abstract: Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

title: Defining the multivalent functions of CTCF from chromatin state and three-dimensional chromatin interactions
abstract: CCCTC-binding factor (CTCF) is a multi-functional protein that is assigned various, even contradictory roles in the genome. High-throughput sequencing-based technologies such as ChIP-seq and Hi-C provided us the opportunity to assess the multivalent functions of CTCF in the human genome. The location of CTCF-binding sites with respect to genomic features provides insights into the possible roles of this protein. Here we present the first genome-wide survey and characterization of three important functions of CTCF: enhancer insulator, chromatin barrier and enhancer linker. We developed a novel computational framework to discover the multivalent functions of CTCF based on chromatin state and three-dimensional chromatin architecture. We applied our method to five human cell lines and identified ∼46 000 non-redundant CTCF sites related to the three functions. Disparate effects of these functions on gene expression were found and distinct genomic features of these CTCF sites were characterized in GM12878 cells. Finally, we investigated the cell-type specificities of CTCF sites related to these functions across five cell types. Our study provides new insights into the multivalent functions of CTCF in the human genome.

title: The three-dimensional architecture of Hox cluster silencing
abstract: Spatial chromatin organization is emerging as an important mechanism to regulate the expression of genes. However, very little is known about genome architecture at high-resolution in vivo. Here, we mapped the three-dimensional organization of the human Hox clusters with chromosome conformation capture (3C) technology. We show that computational modeling of 3C data sets can identify candidate regulatory proteins of chromatin architecture and gene expression. Hox genes encode evolutionarily conserved master regulators of development which strict control has fascinated biologists for over 25 years. Proper transcriptional silencing is key to Hox function since premature expression can lead to developmental defects or human disease. We now show that the HoxA cluster is organized into multiple chromatin loops that are dependent on transcription activity. Long-range contacts were found in all four silent clusters but looping patterns were specific to each cluster. In contrast to the Drosophila homeotic bithorax complex (BX-C), we found that Polycomb proteins are only modestly required for human cluster looping and silencing. However, computational three-dimensional Hox cluster modeling identified the insulator-binding protein CTCF as a likely candidate mediating DNA loops in all clusters. Our data suggest that Hox cluster looping may represent an evolutionarily conserved structural mechanism of transcription regulation.

title: Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders
abstract: Mammalian chromatin is compartmentalized in topologically associating domains (TADs), genomic regions within which sequences preferentially contact each other. This organization has been proposed to be essential to organize the regulatory information contained in mammalian genomes. We show that Six homeobox genes, essential developmental regulators organized in gene clusters across different animal phyla, share a deeply conserved chromatin organization formed by two abutting TADs that predates the Cambrian explosion. This organization is required to generate separate regulatory landscapes for neighboring genes within the cluster, resulting in very different gene expression patterns. Finally, we show that this extremely conserved 3D architecture is associated with a characteristic arrangement of CCCTC-binding factor (CTCF) binding sites in diverging orientations, revealing a genome-wide conserved signature for TAD borders. Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.

title: A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping
abstract: We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.

title: Comparative Hi-C Reveals that CTCF Underlies Evolution of Chromosomal Domain Architecture
abstract: Topological domains are key architectural building blocks of chromosomes, but their functional importance and evolutionary dynamics are not well defined. We performed comparative high-throughput chromosome conformation capture (Hi-C) in four mammals and characterized the conservation and divergence of chromosomal contact insulation and the resulting domain architectures within distantly related genomes. We show that the modular organization of chromosomes is robustly conserved in syntenic regions and that this is compatible with conservation of the binding landscape of the insulator protein CTCF. Specifically, conserved CTCF sites are co-localized with cohesin, are enriched at strong topological domain borders, and bind to DNA motifs with orientations that define the directionality of CTCF’s long-range interactions. Conversely, divergent CTCF binding between species is correlated with divergence of internal domain structure, likely driven by local CTCF binding sequence changes, demonstrating how genome evolution can be linked to a continuous flux of local conformation changes. We also show that large-scale domains are reorganized during genome evolution as intact modules.

title: Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus
abstract: Higher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus. SiRNA-mediated depletion of CTCF or the cohesin component, RAD21, showed that these two factors have distinct roles in regulating the higher order organization of CFTR. CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity. Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs). Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery.

title: 3D Chromosome Regulatory Landscape of Human Pluripotent Cells.
abstract: In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease.