Biology subjects

Lieberman Aiden, E.

Publications and source records attributed to Lieberman Aiden, E..

14 recordsLinked to original sources

bioRxiv2024

Cohesin-mediated 3D contacts tune enhancer-promoter regulation

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes -- those with strong distal enhancers -- are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

molecular biology↗

bioRxiv2022

Proximity-dependent recruitment of Polycomb Repressive Complexes by the lncRNA Airn

During mouse embryogenesis, expression of the lncRNA Airn induces gene silencing and recruits Polycomb Repressive Complexes (PRCs) to varying extents over a 15 megabase domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between Airn lncRNA and target chromatin correlated with underlying intensity of PRC-directed chromatin modifications. Deletion of CpG islands that form contacts with Airn altered long-distance silencing and PRC activity in a manner that correlated with changes in chromatin architecture. We conclude that Airn is a potent cis-acting lncRNA whose primary functions of transcriptional repression and PRC recruitment are controlled by an equilibratory network of DNA regulatory elements that modulate its frequency of contact with target chromatin.

genomics↗

bioRxiv2021

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface.

The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesins LE during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells from patients carrying mutations in the Mcph1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin IIs NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1s ability to block condensin IIs association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both SMC complexes must be tightly regulated to adjust both the chromatids structure and their segregation.

cell biology↗

bioRxiv2019

Chromosomal-level genome assembly of the scimitar-horned oryx: insights into diversity and demography of a species extinct in the wild

Captive populations provide a valuable insurance against extinctions in the wild. However, they are also vulnerable to the negative impacts of inbreeding, selection and drift. Genetic information is therefore considered a critical aspect of conservation management planning. Recent developments in sequencing technologies have the potential to improve the outcomes of management programmes however, the transfer of these approaches to applied conservation has been slow. The scimitar-horned oryx (Oryx dammah) is a North African antelope that has been extinct in the wild since the early 1980s and is the focus of a long-term reintroduction project. To enable the selection of suitable founder individuals, facilitate post-release monitoring and improve captive breeding management, comprehensive genomic resources are required. Here, we used 10X Chromium sequencing together with Hi-C contact mapping to develop a chromosomal-level genome assembly for the species. The resulting assembly contained 29 chromosomes with a scaffold N50 of 100.4 Mb, and displayed strong chromosomal synteny with the cattle genome. Using resequencing data from six additional individuals, we demonstrated relatively high genetic diversity in the scimitar-horned oryx compared to other mammals, despite it having experienced a strong founding event in captivity. Additionally, the level of diversity across populations varied according to management strategy. Finally, we uncovered a dynamic demographic history that coincided with periods of climate variation during the Pleistocene. Overall, our study provides a clear example of how genomic data can uncover valuable insights into captive populations and contributes important resources to guide future management decisions of an endangered species.

genomics↗

bioRxiv2019

Cohesin depleted cells pass through mitosis and reconstitute a functional nuclear architecture

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is debatable. Using live-cell and super-resolved 3D microscopy, we demonstrate that cohesin depleted cells pass through an endomitosis and rebuild a single multilobulated nucleus (MLN) with chromosome territories (CTs) pervaded by interchromatin channels. CTs contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. Splicing speckles are located nearby within the lining channel system. These clusters form microscopically defined, active and inactive compartments, which correspond to A/B compartments, detected with ensemble Hi-C. Functionality of MLN despite continuous absence of cohesin was demonstrated by their ability to pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.

cell biology↗

bioRxiv2019

Activity-by-Contact model of enhancer specificity from thousands of CRISPR perturbations

Mammalian genomes harbor millions of noncoding elements called enhancers that quantitatively regulate gene expression, but it remains unclear which enhancers regulate which genes. Here we describe an experimental approach, based on CRISPR interference, RNA FISH, and flow cytometry (CRISPRi-FlowFISH), to perturb enhancers in the genome, and apply it to test >3,000 potential regulatory enhancer-gene connections across multiple genomic loci. A simple equation based on a mechanistic model for enhancer function performed remarkably well at predicting the complex patterns of regulatory connections we observe in our CRISPR dataset. This Activity-by-Contact (ABC) model involves multiplying measures of enhancer activity and enhancer-promoter 3D contacts, and can predict enhancer-gene connections in a given cell type based on chromatin state maps. Together, CRISPRi-FlowFISH and the ABC model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.

genetics↗

bioRxiv2018

CTCF looping is established during gastrulation in medaka embryos

AbstractGenome architecture plays a critical role in gene regulation, but how the structures seen in mature cells emerge during embryonic development remains poorly understood. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation which is the most dramatic event in early embryogenesis, and characterize transcription, protein binding, and genome architecture. We find that gastrulation is most associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and great increase in the size of contact domains. However, the position of CTCF is fixed throughout medaka embryogenesis. Interestingly, genome-wide transcription precedes the emergence of mature domains and CTCF-CTCF loops.

genomics↗

bioRxiv2018

Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

Chromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb.\n\nOne Sentence SummarySuper-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.

genetics↗

bioRxiv2017

A Cell Type-Specific Class of Chromatin Loops Anchored at Large DNA Methylation Nadirs

Higher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we found that large (~10kb) DNA methylation nadirs can form long loops connecting anchor loci that may be dozens of megabases apart, as well as interchromosomal links. The interacting loci comprise ~3.5Mb of the human genome. The data are more consistent with the formation of these loops by phase separation of the interacting loci to form a genomic subcompartment, rather than with CTCF-mediated extrusion. Interestingly, unlike previously characterized genomic subcompartments, this subcompartment is only present in particular cell types, such as stem and progenitor cells. Further, we identify one particular loop anchor that is functionally associated with maintenance of the hematopoietic stem cell state. Our work reveals that H3K27me3-marked large DNA methylation nadirs represent a novel set of very long-range loops and links associated with cellular identity.\n\nSummaryHi-C and DNA methylation analyses reveal novel chromatin loops between distant sites implicated in stem and progenitor cell function.

genomics↗

bioRxiv2017

De Novo Prediction of Human Chromosome Structures: Epigenetic Marking Patterns Encode Genome Architecture

Inside the cell nucleus, genomes fold into organized structures that are characteristic of cell type. Here, we show that this chromatin architecture can be predicted de novo using epigenetic data derived from ChIP-Seq. We exploit the idea that chromosomes encode a one-dimensional sequence of chromatin structural types. Interactions between these chromatin types determine the three-dimensional (3D) structural ensemble of chromosomes through a process similar to phase separation. First, a recurrent neural network is used to infer the relation between the epigenetic marks present at a locus, as assayed by ChIP-Seq, and the genomic compartment in which those loci reside, as measured by DNA-DNA proximity ligation (Hi-C). Next, types inferred from this neural network are used as an input to an energy landscape model for chromatin organization (MiChroM) in order to generate an ensemble of 3D chromosome conformations. After training the model, dubbed MEGABASE (Maximum Entropy Genomic Annotation from Biomarkers Associated to Structural Ensembles), on odd numbered chromosomes, we predict the chromatin type sequences and the subsequent 3D conformational ensembles for the even chromosomes. We validate these structural ensembles by using ChIP-Seq tracks alone to predict Hi-C maps as well as distances measured using 3D FISH experiments. Both sets of experiments support the hypothesis of phase separation being the driving process behind compartmentalization. These findings strongly suggest that epigenetic marking patterns encode sufficient information to determine the global architecture of chromosomes and that de novo structure prediction for whole genomes may be increasingly possible.

biophysics↗

bioRxiv2017

Static And Dynamic DNA Loops Form AP-1 Bound Activation Hubs During Macrophage Development

The three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops during human monocyte-to-macrophage differentiation. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer-activation of preformed loops represent two distinct modes of regulation that together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for Activator Protein 1 (AP-1) binding events, suggesting multi-loop activation hubs driven by cell-type specific transcription factors may represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.\n\nHIGHLIGHTSO_LIHigh resolution and high sensitivity of loop detection via deeply sequenced in situ Hi-C experiments during monocyte to macrophage differentiation (> 10 billion total reads)\nC_LIO_LIMulti-loop interaction communities identified surrounding key macrophage genes.\nC_LIO_LIMulti-loop communities connect dynamic enhancers through both static and newly acquired DNA loops, forming hubs of activation\nC_LIO_LIMacrophage activation hubs are enriched for AP-1 bound long-range enhancer interactions, suggesting cell-type specific TFs drive changes in 3D structure and transcription through regulatory DNA loops\nC_LI

genomics↗

bioRxiv2017

Cohesin Loss Eliminates All Loop Domains, Leading To Links Among Superenhancers And Downregulation Of Nearby Genes

The human genome folds to create thousands of intervals, called \"contact domains,\" that exhibit enhanced contact frequency within themselves. \"Loop domains\" form because of tethering between two loci - almost always bound by CTCF and cohesin - lying on the same chromosome. \"Compartment domains\" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loci in different compartments that had been in the same loop domain become more segregated. Loss of loop domains does not lead to widespread ectopic gene activation, but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes, and affecting the regulation of nearby genes. Cohesin restoration quickly reverses these effects, consistent with a model where loop extrusion is rapid.

genomics↗

bioRxiv2017

Polycomb-Mediated Chromatin Loops Revealed by a Sub-Kilobase Resolution Chromatin Interaction Map

The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or \"anchors\" are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of Polycomb Repressive Complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, our results indicate that loops are also associated with gene repression.

molecular biology↗

bioRxiv2016

Genetic determinants of chromatin accessibility and gene regulation in T cell activation across human individuals

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) and RNA-seq profiles from activated CD4+ T cells of up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, in patterns consistent with the 3D organization of chromosomes measured by in situ Hi-C in T cells. 15% of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak through disrupting binding sites for transcription factors important for T cell differentiation and activation. These ATAC quantitative trait nucleotides (ATAC-QTNs) have the largest effects on co-accessible peaks, are associated with gene expression from the same aliquot of cells, are rarely affecting core binding motifs, and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis- regulatory elements, in isolation or in concert, to influence gene expression in primary immune cells that play a key role in many human diseases.

genomics↗