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Researchers in the Simmons lab chart the embryonic origins and appearance of diverse intestinal cellular compartments, with important implications for intestinal diseases.
Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.
Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.
Reactivation of a developmentally silenced embryonic globin gene.
The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.
Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture.
Chromosome conformation capture (3C) methods measure the spatial proximity between DNA elements in the cell nucleus. Many methods have been developed to sample 3C material, including the Capture-C family of protocols. Capture-C methods use oligonucleotides to enrich for interactions of interest from sequencing-ready 3C libraries. This approach is modular and has been adapted and optimized to work for sampling of disperse DNA elements (NuTi Capture-C), including from low cell inputs (LI Capture-C), as well as to generate Hi-C like maps for specific regions of interest (Tiled-C) and to interrogate multiway interactions (Tri-C). We present the design, experimental protocol and analysis pipeline for NuTi Capture-C in addition to the variations for generation of LI Capture-C, Tiled-C and Tri-C data. The entire procedure can be performed in 3 weeks and requires standard molecular biology skills and equipment, access to a next-generation sequencing platform, and basic bioinformatic skills. Implemented with other sequencing technologies, these methods can be used to identify regulatory interactions and to compare the structural organization of the genome in different cell types and genetic models.
MLL-AF4 cooperates with PAF1 and FACT to drive high-density enhancer interactions in leukemia.
Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.
The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes.
Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.
BET inhibition disrupts transcription but retains enhancer-promoter contact
Summary In higher eukaryotes, enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. The formation of phase condensates is thought to be an essential component of enhancer function. Here, we show that pharmacological targeting of cells with inhibitors of BET (Bromodomain and Extra-Terminal domain) proteins can have a strong impact on transcription but very little impact on enhancer-promoter interactions. Treatment with 1,6-hexanediol, which dissolves phase condensate structures and reduces BET and Mediator protein binding at enhancers, can also have a strong effect on gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further suggest that enhancer-promoter interactions are not dependent on high levels of BRD4 (Bromodomain-containing protein 4) and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and loop extrusion by CTCF/cohesin.
Genetic dissection of the α-globin super-enhancer in vivo.
Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.
Multiplexed analysis of chromosome conformation at vastly improved sensitivity.
Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.
How best to identify chromosomal interactions: a comparison of approaches.
Chromosome conformation capture (3C) methods are central to understanding the link between nuclear structure and function, and the physical interactions between distal regulatory elements and promoters. However, no one method is appropriate to address all biological questions, as each variant differs markedly in resolution, reproducibility, throughput and biases. A thorough appreciation of the strengths and weaknesses of each technique is critical when choosing the correct method for a specific application or for gauging how best to interpret different sources of data. In addition, the analysis method must be carefully considered, as this choice can profoundly affect the output. In this Review, we describe and compare the different available 3C-based approaches, with a focus on the analysis of mammalian genomes.
CaptureCompendium: a comprehensive toolkit for 3C analysis
ABSTRACT DNA folding within nuclei is a highly ordered process, with implications for gene regulation and development. An array of chromosome conformation capture (3C) methods have been developed to investigate how DNA is packaged within nuclei and to interrogate specific interactions. While these methods use different approaches to examine target loci (many-versus-all) or the entire genome (all-versus-all), they all rely on the core principle of endonuclease digestion and proximity-based ligation to re-arrange genomic order to reflect the three-dimensional nuclear conformation. This sequence reorganization creates novel chimeric DNA fragments which require specialist bioinformatic tools to analyze and visualize. Despite this need for specialist bioinformatic skills, the core biological importance of genome folding has seen widespread methodological uptake. To service the needs of experimentalists using the many-versus-all Capture-C family of methods we have developed CaptureCompendium; a toolkit of software to simplify the design, analysis and presentation of 3C experiments.
Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains.
The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.
Identification of LZTFL1 as a candidate effector gene at a COVID-19 risk locus.
The severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) disease (COVID-19) pandemic has caused millions of deaths worldwide. Genome-wide association studies identified the 3p21.31 region as conferring a twofold increased risk of respiratory failure. Here, using a combined multiomics and machine learning approach, we identify the gain-of-function risk A allele of an SNP, rs17713054G>A, as a probable causative variant. We show with chromosome conformation capture and gene-expression analysis that the rs17713054-affected enhancer upregulates the interacting gene, leucine zipper transcription factor like 1 (LZTFL1). Selective spatial transcriptomic analysis of lung biopsies from patients with COVID-19 shows the presence of signals associated with epithelial-mesenchymal transition (EMT), a viral response pathway that is regulated by LZTFL1. We conclude that pulmonary epithelial cells undergoing EMT, rather than immune cells, are likely responsible for the 3p21.31-associated risk. Since the 3p21.31 effect is conferred by a gain-of-function, LZTFL1 may represent a therapeutic target.
Targeted high-resolution chromosome conformation capture at genome-wide scale
ABSTRACT Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at up to several hundred loci. All 3C methods are affected to varying degrees by inefficiency, bias and noise. As such, generation of reproducible high-resolution interaction profiles has not been achieved at scale. To overcome this barrier, we systematically tested and improved upon current methods. We show that isolation of 3C libraries from intact nuclei, as well as shortening and titration of enrichment oligonucleotides used in high-resolution methods reduces noise and increases on-target sequencing. We combined these technical modifications into a new method Nuclear-Titrated (NuTi) Capture-C, which provides a >3-fold increase in informative sequencing content over current Capture-C protocols. Using NuTi Capture-C we target 8,061 promoters in triplicate, demonstrating that this method generates reproducible high-resolution genome-wide 3C interaction profiles at scale.
Mapping chromatin structure at base-pair resolution unveils a unified model of cis-regulatory element interactions.
Chromatin structure is a key determinant of gene expression in eukaryotes, but it has not been possible to define the structure of cis-regulatory elements at the scale of the proteins that bind them. Here, we generate multidimensional chromosome conformation capture (3C) maps at base-pair resolution using Micro Capture-C ultra (MCCu). This can resolve contacts between individual transcription factor motifs within cis-regulatory elements. Using degron systems, we show that removal of Mediator complex components alters fine-scale promoter structure and that nucleosome depletion plays a key role in transcription factor-driven enhancer-promoter contacts. We observe that chromatin is partitioned into nanoscale domains by nucleosome-depleted regions. This structural conformation is reproduced by chemically specific coarse-grained molecular dynamics simulations of the physicochemical properties of chromatin. Combining MCCu with molecular dynamics simulations and super-resolution microscopy allows us to propose a unified model in which the biophysical properties of chromatin orchestrate contacts between cis-regulatory elements.
Ancient genomic linkage of α-globin and Nprl3 couples metabolism with erythropoiesis.
Red blood cell development from erythroid progenitors requires profound reshaping of metabolism and gene expression. How these transcriptional and metabolic alterations are coupled is unclear. Nprl3 (an inhibitor of mTORC1) has remained in synteny with the α-globin genes for >500 million years, and harbours most of the a-globin enhancers. However, whether Nprl3 serves an erythroid role is unknown. We found that while haematopoietic progenitors require basal Nprl3 expression, erythroid Nprl3 expression is further boosted by the α-globin enhancers. This lineage-specific upregulation is required for sufficient erythropoiesis. Loss of Nprl3 affects erythroblast metabolism via elevating mTORC1 signalling, suppressing autophagy and disrupting glycolysis. Broadly consistent with these murine findings, human NPRL3-knockout erythroid progenitors produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Therefore, we propose that the anciently conserved linkage of NprI3, α-globin and their associated enhancers has coupled metabolic and developmental control of erythropoiesis.