an-integrated-workflow-to-study-promoter-centric-spatio-temporal

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Josep Carreras Leukaemia Research Institute

Institute for Health Science Research Germans Trias i Pujol

Long-range chromosomal interactions bring distal regulatory elements and promoters together to regulate gene expression in biological processes. By performing promoter capture Hi-C (PCHi-C) on human embryonic stem cell-derived cardiomyocytes (hESC-CMs), we show that such promoter interactions are a key mechanism by which enhancers contact their target genes after hESC-CM differentiation from hESCs. We also show that the promoter interactome of hESC-CMs is associated with expression quantitative trait loci (eQTLs) in cardiac left ventricular tissue; captures the dynamic process of genome reorganisation after hESC-CM differentiation; overlaps genome-wide association study (GWAS) regions associated with heart rate; and identifies new candidate genes in such regions. These findings indicate that regulatory elements in hESC-CMs identified by our approach control gene expression involved in ventricular conduction and rhythm of the heart. The study of promoter interactions in other hESC-derived cell types may be of utility in functional investigation of GWAS-associated regions.

Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.

In the original version of the Article, the gene symbol for tissue factor pathway inhibitor was inadvertently given as 'TFP1' instead of 'TFPI'. This has now been corrected in both the PDF and HTML versions of the Article.

Author Correction: Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.

Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer clusters or super-enhancers. So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in three-dimensional (3D) space. Furthermore, their target genes are often unknown. We have created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers to their target genes, often located hundreds of kilobases away. It also revealed >1,300 groups of islet enhancers, super-enhancers and active promoters that form 3D hubs, some of which show coordinated glucose-dependent activity. We demonstrate that genetic variation in hubs impacts insulin secretion heritability, and show that hub annotations can be used for polygenic scores that predict T2D risk driven by islet regulatory variants. Human islet 3D chromatin architecture, therefore, provides a framework for interpretation of T2D genome-wide association study (GWAS) signals.

Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes.

B-cell leukemia transdifferentiation to macrophage involves reconfiguration of DNA methylation for long-range regulation.

B lymphopoiesis is tightly regulated at the level of gene transcription. In recent years, investigators have shed light on the transcription factor networks and the epigenetic machinery involved at all differentiation steps of mammalian B cell development. During terminal differentiation, B cells undergo dramatic changes in gene transcriptional programs to generate germinal center B cells, plasma cells and memory B cells. Recent evidence indicates that mature B cell formation involves an essential contribution from 3D chromatin conformations through its interplay with transcription factors and epigenetic machinery. Here, we provide an up-to-date overview of the coordination between transcription factors, epigenetic changes, and chromatin architecture during terminal B cell differentiation, focusing on recent discoveries and technical advances for studying 3D chromatin structures.

From Loops to Looks: Transcription Factors and Chromatin Organization Shaping Terminal B Cell Differentiation.

Adipocyte differentiation is driven by waves of transcriptional regulators that reprogram the enhancer landscape and change the wiring of the promoter interactome. Here, we use high-throughput chromosome conformation enhancer capture to interrogate the role of enhancer-to-enhancer interactions during differentiation of human mesenchymal stem cells. We find that enhancers form an elaborate network that is dynamic during differentiation and coupled with changes in enhancer activity. Transcription factors (TFs) at baited enhancers amplify TF binding at target enhancers, a phenomenon we term cross-interaction stabilization of TFs. Moreover, highly interconnected enhancers (HICE) act as integration hubs orchestrating differentiation by the formation of three-dimensional enhancer communities, inside which, HICE, and other enhancers, converge on phenotypically important gene promoters. Collectively, these results indicate that enhancer interactions play a key role in the regulation of enhancer function, and that HICE are important for both signal integration and compartmentalization of the genome.

Highly interconnected enhancer communities control lineage-determining genes in human mesenchymal stem cells.

Associations between blood cancer and genetic predisposition, including both inherited variants and acquired mutations and epimutations, have been well characterized. However, the majority of these variants affect noncoding regions, making their mechanisms difficult to hypothesize and hindering the translation of these insights into patient benefits. Fueled by unprecedented progress in next-generation sequencing and computational integrative analysis, studies have started applying combinations of epigenetic, genome architecture, and functional assays to bridge the gap between noncoding variants and blood cancer. These complementary tools have not only allowed us to understand the potential malignant role of these variants but also to differentiate key variants, cell-types, and conditions from misleading ones. Here, we briefly review recent studies that have provided fundamental insights into our understanding of how noncoding mutations at enhancers predispose and promote blood malignancies in the context of spatial genome architecture.

The Genome in a Three-Dimensional Context: Deciphering the Contribution of Noncoding Mutations at Enhancers to Blood Cancer.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.

Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease.

The MYC axis is disrupted in cancer, predominantly through activation of the MYC family oncogenes but also through inactivation of the MYC partner MAX or of the MAX partner MGA. MGA and MAX are also members of the polycomb repressive complex, ncPRC1.6. Here, we use genetically modified MAX-deficient small-cell lung cancer (SCLC) cells and carry out genome-wide and proteomics analyses to study the tumor suppressor function of MAX. We find that MAX mutant SCLCs have ASCL1 or NEUROD1 or combined ASCL1/NEUROD1 characteristics and lack MYC transcriptional activity. MAX restitution triggers prodifferentiation expression profiles that shift when MAX and oncogenic MYC are coexpressed. Although ncPRC1.6 can be formed, the lack of MAX restricts global MGA occupancy, selectively driving its recruitment toward E2F6-binding motifs. Conversely, MAX restitution enhances MGA occupancy to repress genes involved in different functions, including stem cell and DNA repair/replication. Collectively, these findings reveal that MAX mutant SCLCs have either ASCL1 or NEUROD1 or combined characteristics and are MYC independent and exhibit deficient ncPRC1.6-mediated gene repression.

MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex.

Low-flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3ca activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low-flow vascular malformations.

The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib.

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to 3D genome structure remains poorly understood. Here, we used the highly predictive heteromorphic polymer (HiP-HoP) model to predict chromatin conformations at the proto-oncogene  in healthy and malignant B cells. After confirming that the model gives good predictions of Hi-C data for the nonmalignant human B cell-derived cell line GM12878, we generated predictions for two cancer cell lines, U266 and Z-138. These possess genome rearrangements involving  and the immunoglobulin heavy locus (), which we mapped using targeted genome sequencing. Our simulations showed that a rearrangement in U266 cells where a single  super-enhancer is inserted next to  leaves the local topologically associated domain (TAD) structure intact. We also observed extensive changes in enhancer-promoter interactions within the TAD, suggesting that it is the downstream chromatin remodeling which gives rise to the oncogene activation, rather than the presence of the inserted super-enhancer DNA sequence per se. Simulations of the - reciprocal translocation in Z-138 cells revealed that an oncogenic fusion TAD is created, encompassing  and the  super-enhancers. We predicted how the structure and expression of  changes in these different cell lines, validating this using qPCR and fluorescence in situ hybridization microscopy. Our work demonstrates the power of polymer simulations to predict differences in chromatin interactions and gene expression for different translocation breakpoints.

High-resolution simulations of chromatin folding at genomic rearrangements in malignant B cells provide mechanistic insights into proto-oncogene deregulation.

Correct B cell identity at each stage of cellular differentiation during B lymphocyte development is critically dependent on a tightly controlled epigenomic landscape. We previously identified HDAC7 as an essential regulator of early B cell development and its absence leads to a drastic block at the pro-B to pre-B cell transition. More recently, we demonstrated that HDAC7 loss in pro-B-ALL in infants associates with a worse prognosis. Here we delineate the molecular mechanisms by which HDAC7 modulates early B cell development. We find that HDAC7 deficiency drives global chromatin de-condensation, histone marks deposition and deregulates other epigenetic regulators and mobile elements. Specifically, the absence of HDAC7 induces TET2 expression, which promotes DNA 5-hydroxymethylation and chromatin de-condensation. HDAC7 deficiency also results in the aberrant expression of microRNAs and LINE-1 transposable elements. These findings shed light on the mechanisms by which HDAC7 loss or misregulation may lead to B cell-based hematological malignancies.

The HDAC7-TET2 epigenetic axis is essential during early B lymphocyte development.

Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis.

Article	Year
Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks. Nature communications\| PubMed ID: 29955040	2018
Author Correction: Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks. Nature communications\| PubMed ID: 30420621	2018
Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes. Nature genetics\| PubMed ID: 31253982	2019
B-cell leukemia transdifferentiation to macrophage involves reconfiguration of DNA methylation for long-range regulation. Leukemia\| PubMed ID: 31719676	2020
From Loops to Looks: Transcription Factors and Chromatin Organization Shaping Terminal B Cell Differentiation. Trends in immunology\| PubMed ID: 31822368	2020
Highly interconnected enhancer communities control lineage-determining genes in human mesenchymal stem cells. Nature genetics\| PubMed ID: 33020665	2020
The Genome in a Three-Dimensional Context: Deciphering the Contribution of Noncoding Mutations at Enhancers to Blood Cancer. Frontiers in immunology\| PubMed ID: 33117405	2020
Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease. Nature communications\| PubMed ID: 33863903	2021
MAX mutant small-cell lung cancers exhibit impaired activities of MGA-dependent noncanonical polycomb repressive complex. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34493659	2021
The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib. EMBO molecular medicine\| PubMed ID: 35695059	2022
High-resolution simulations of chromatin folding at genomic rearrangements in malignant B cells provide mechanistic insights into proto-oncogene deregulation. Genome research\| PubMed ID: 35863900	2022
The HDAC7-TET2 epigenetic axis is essential during early B lymphocyte development. Nucleic acids research\| PubMed ID: 35904805	2022
Low input capture Hi-C (liCHi-C) identifies promoter-enhancer interactions at high-resolution. Nature communications\| PubMed ID: 36650138	2023

Biola M. Javierre

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