Publications by authors named "Job Dekker"

169 Publications

Inner nuclear protein Matrin-3 coordinates cell differentiation by stabilizing chromatin architecture.

Nat Commun 2021 10 29;12(1):6241. Epub 2021 Oct 29.

Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute (DFCI), Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA.

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation.
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http://dx.doi.org/10.1038/s41467-021-26574-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8556400PMC
October 2021

Mechanisms of Chromosome Folding and Nuclear Organization: Their Interplay and Open Questions.

Cold Spring Harb Perspect Biol 2021 Sep 13. Epub 2021 Sep 13.

Howard Hughes Medical Institute, and Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Microscopy and genomic approaches provide detailed descriptions of the three-dimensional folding of chromosomes and nuclear organization. The fundamental question is how activity of molecules at the nanometer scale can lead to complex and orchestrated spatial organization at the scale of chromosomes and the whole nucleus. At least three key mechanisms can bridge across scales: (1) tethering of specific loci to nuclear landmarks leads to massive reorganization of the nucleus; (2) spatial compartmentalization of chromatin, which is driven by molecular affinities, results in spatial isolation of active and inactive chromatin; and (3) loop extrusion activity of SMC (structural maintenance of chromosome) complexes can explain many features of interphase chromatin folding and underlies key phenomena during mitosis. Interestingly, many features of chromosome organization ultimately result from collective action and the interplay between these mechanisms, and are further modulated by transcription and topological constraints. Finally, we highlight some outstanding questions that are critical for our understanding of nuclear organization and function. We believe many of these questions can be answered in the coming years.
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http://dx.doi.org/10.1101/cshperspect.a040147DOI Listing
September 2021

Symbiodinium microadriaticum (coral microalgal endosymbiont).

Trends Genet 2021 Nov 11;37(11):1044-1045. Epub 2021 Sep 11.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. Electronic address:

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http://dx.doi.org/10.1016/j.tig.2021.08.008DOI Listing
November 2021

Systematic evaluation of chromosome conformation capture assays.

Nat Methods 2021 09 3;18(9):1046-1055. Epub 2021 Sep 3.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.
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http://dx.doi.org/10.1038/s41592-021-01248-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446342PMC
September 2021

Linker histone H1.8 inhibits chromatin binding of condensins and DNA topoisomerase II to tune chromosome length and individualization.

Elife 2021 08 18;10. Epub 2021 Aug 18.

Laboratory of Chromosome and Cell Biology, The Rockefeller University, New York, United States.

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.
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http://dx.doi.org/10.7554/eLife.68918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416026PMC
August 2021

Hi-C 3.0: Improved Protocol for Genome-Wide Chromosome Conformation Capture.

Curr Protoc 2021 Jul;1(7):e198

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts.

The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a chromosome conformation capture (3C)-based technology to detect pair-wise chromatin interactions genome-wide, and has become a benchmark tool to study genome organization. In Hi-C, chromatin conformation is first captured by chemical cross-linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high-throughput sequencing, which enables the mapping of pair-wise chromatin interactions genome-wide. Over the past decade, "next-generation" sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in-depth guide to performing an up-to-date Hi-C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross-linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi-C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi-C sequencing library preparation.
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http://dx.doi.org/10.1002/cpz1.198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8362010PMC
July 2021

Chromosome-Level Assembly of the Atlantic Silverside Genome Reveals Extreme Levels of Sequence Diversity and Structural Genetic Variation.

Genome Biol Evol 2021 06;13(6)

Department of Natural Resources, Cornell University, Ithaca, New York, USA.

The levels and distribution of standing genetic variation in a genome can provide a wealth of insights about the adaptive potential, demographic history, and genome structure of a population or species. As structural variants are increasingly associated with traits important for adaptation and speciation, investigating both sequence and structural variation is essential for wholly tapping this potential. Using a combination of shotgun sequencing, 10x Genomics linked reads and proximity-ligation data (Chicago and Hi-C), we produced and annotated a chromosome-level genome assembly for the Atlantic silverside (Menidia menidia)-an established ecological model for studying the phenotypic effects of natural and artificial selection-and examined patterns of genomic variation across two individuals sampled from different populations with divergent local adaptations. Levels of diversity varied substantially across each chromosome, consistently being highly elevated near the ends (presumably near telomeric regions) and dipping to near zero around putative centromeres. Overall, our estimate of the genome-wide average heterozygosity in the Atlantic silverside is among the highest reported for a fish, or any vertebrate (1.32-1.76% depending on inference method and sample). Furthermore, we also found extreme levels of structural variation, affecting ∼23% of the total genome sequence, including multiple large inversions (> 1 Mb and up to 12.6 Mb) associated with previously identified haploblocks showing strong differentiation between locally adapted populations. These extreme levels of standing genetic variation are likely associated with large effective population sizes and may help explain the remarkable adaptive divergence among populations of the Atlantic silverside.
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http://dx.doi.org/10.1093/gbe/evab098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214408PMC
June 2021

Genetic and spatial organization of the unusual chromosomes of the dinoflagellate Symbiodinium microadriaticum.

Nat Genet 2021 05 29;53(5):618-629. Epub 2021 Apr 29.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

Dinoflagellates are main primary producers in the oceans, the cause of algal blooms and endosymbionts of marine invertebrates. Much remains to be understood about their biology, including their peculiar crystalline chromosomes. We assembled 94 chromosome-scale scaffolds of the genome of the coral endosymbiont Symbiodinium microadriaticum and analyzed their organization. Genes are enriched towards the ends of chromosomes and are arranged in alternating unidirectional blocks. Some chromosomes are enriched for genes involved in specific biological processes. The chromosomes fold as linear rods and each is composed of a series of structural domains separated by boundaries. Domain boundaries are positioned at sites where transcription of two gene blocks converges and disappear when cells are treated with chemicals that block transcription, indicating correlations between gene orientation, transcription and chromosome folding. The description of the genetic and spatial organization of the S. microadriaticum genome provides a foundation for deeper exploration of the extraordinary biology of dinoflagellates and their chromosomes.
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http://dx.doi.org/10.1038/s41588-021-00841-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110479PMC
May 2021

Liquid chromatin Hi-C characterizes compartment-dependent chromatin interaction dynamics.

Nat Genet 2021 03 11;53(3):367-378. Epub 2021 Feb 11.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10-25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.
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http://dx.doi.org/10.1038/s41588-021-00784-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946813PMC
March 2021

Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2-mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
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http://dx.doi.org/10.1172/jci.insight.142149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934867PMC
February 2021

Is Involved in Corneal Stroma Development by Regulating Neural Crest Migration.

Int J Mol Sci 2020 Oct 21;21(20). Epub 2020 Oct 21.

Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China.

Previously, we identified RAD21 from a peripheral sclerocornea pedigree. Injection of this variant mRNA into embryos disrupted the organization of corneal stroma fibrils. To understand the mechanisms of RAD21-mediated corneal stroma defects, gene expression and chromosome conformation analysis were performed using cells from family members affected by peripheral sclerocornea. Both gene expression and chromosome conformation of cell adhesion genes were affected in cells carrying the heterozygous variant. Since cell migration is essential in early embryonic development and sclerocornea is a congenital disease, we studied neural crest migration during cornea development in embryos. In embryos injected with mutant mRNA, neural crest migration was disrupted, and the number of neural crest-derived periocular mesenchymes decreased significantly in the corneal stroma region. Our data indicate that the RAD21 variant contributes to peripheral sclerocornea by modifying chromosome conformation and gene expression, therefore disturbing neural crest cell migration, which suggests RAD21 plays a key role in corneal stroma development.
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http://dx.doi.org/10.3390/ijms21207807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594026PMC
October 2020

Detecting chromatin interactions between and along sister chromatids with SisterC.

Nat Methods 2020 10 23;17(10):1002-1009. Epub 2020 Sep 23.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2'-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis.
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http://dx.doi.org/10.1038/s41592-020-0930-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541687PMC
October 2020

Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain.

EMBO J 2020 11 16;39(21):e99520. Epub 2020 Sep 16.

CNRS, Institut Jacques Monod, Equipe Labellisée Association Pour la Recherche sur le Cancer, Université de Paris, Paris, France.

Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains.
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http://dx.doi.org/10.15252/embj.201899520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604622PMC
November 2020

Multi-contact 3C reveals that the human genome during interphase is largely not entangled.

Nat Struct Mol Biol 2020 12 14;27(12):1105-1114. Epub 2020 Sep 14.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

During interphase, the eukaryotic genome is organized into chromosome territories that are spatially segregated into compartment domains. The extent to which interacting domains or chromosomes are entangled is not known. We analyze series of co-occurring chromatin interactions using multi-contact 3C (MC-3C) in human cells to provide insights into the topological entanglement of chromatin. Multi-contact interactions represent percolation paths (C-walks) through three-dimensional (3D) chromatin space. We find that the order of interactions within C-walks that occur across interfaces where chromosomes or compartment domains interact is not random. Polymer simulations show that such C-walks are consistent with distal domains being topologically insulated, that is, not catenated. Simulations show that even low levels of random strand passage, for example by topoisomerase II, would result in entanglements, increased mixing at domain interfaces and an order of interactions within C-walks not consistent with experimental MC-3C data. Our results indicate that, during interphase, entanglements between chromosomes and chromosomal domains are rare.
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http://dx.doi.org/10.1038/s41594-020-0506-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718335PMC
December 2020

Large domains of heterochromatin direct the formation of short mitotic chromosome loops.

Elife 2020 09 11;9. Epub 2020 Sep 11.

Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.
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http://dx.doi.org/10.7554/eLife.57212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515631PMC
September 2020

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

Nature 2020 07 29;583(7818):699-710. Epub 2020 Jul 29.

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE and Roadmap Epigenomics data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
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http://dx.doi.org/10.1038/s41586-020-2493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410828PMC
July 2020

Mechanisms and Functions of Chromosome Compartmentalization.

Trends Biochem Sci 2020 05 18;45(5):385-396. Epub 2020 Feb 18.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

Active and inactive chromatin are spatially separated in the nucleus. In Hi-C data, this is reflected by the formation of compartments, whose interactions form a characteristic checkerboard pattern in chromatin interaction maps. Only recently have the mechanisms that drive this separation come into view. Here, we discuss new insights into these mechanisms and possible functions in genome regulation. Compartmentalization can be understood as a microphase-segregated block co-polymer. Microphase separation can be facilitated by chromatin factors that associate with compartment domains, and that can engage in liquid-liquid phase separation to form subnuclear bodies, as well as by acting as bridging factors between polymer sections. We then discuss how a spatially segregated state of the genome can contribute to gene regulation, and highlight experimental challenges for testing these structure-function relationships.
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http://dx.doi.org/10.1016/j.tibs.2020.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275117PMC
May 2020

Ultrastructural Details of Mammalian Chromosome Architecture.

Mol Cell 2020 05 25;78(3):554-565.e7. Epub 2020 Mar 25.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of ∼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.
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http://dx.doi.org/10.1016/j.molcel.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222625PMC
May 2020

SPEN integrates transcriptional and epigenetic control of X-inactivation.

Nature 2020 02 5;578(7795):455-460. Epub 2020 Feb 5.

European Molecular Biology Laboratory, Director's Unit, Heidelberg, Germany.

Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN, the loss of which has been associated with deficient XCI at multiple loci. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the mA RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.
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http://dx.doi.org/10.1038/s41586-020-1974-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035112PMC
February 2020

Transcriptional Silencers in Drosophila Serve a Dual Role as Transcriptional Enhancers in Alternate Cellular Contexts.

Mol Cell 2020 01 5;77(2):324-337.e8. Epub 2019 Nov 5.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA 02138, USA; Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. Here, we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that we identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. Our results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and 10-10 in humans.
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http://dx.doi.org/10.1016/j.molcel.2019.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980774PMC
January 2020

A chromosome folding intermediate at the condensin-to-cohesin transition during telophase.

Nat Cell Biol 2019 11 4;21(11):1393-1402. Epub 2019 Nov 4.

Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

Chromosome folding is modulated as cells progress through the cell cycle. During mitosis, condensins fold chromosomes into helical loop arrays. In interphase, the cohesin complex generates loops and topologically associating domains (TADs), while a separate process of compartmentalization drives segregation of active and inactive chromatin. We used synchronized cell cultures to determine how the mitotic chromosome conformation transforms into the interphase state. Using high-throughput chromosome conformation capture (Hi-C) analysis, chromatin binding assays and immunofluorescence, we show that, by telophase, condensin-mediated loops are lost and a transient folding intermediate is formed that is devoid of most loops. By cytokinesis, cohesin-mediated CTCF-CTCF loops and the positions of TADs emerge. Compartment boundaries are also established early, but long-range compartmentalization is a slow process and proceeds for hours after cells enter G1. Our results reveal the kinetics and order of events by which the interphase chromosome state is formed and identify telophase as a critical transition between condensin- and cohesin-driven chromosome folding.
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http://dx.doi.org/10.1038/s41556-019-0406-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858582PMC
November 2019

Highly structured homolog pairing reflects functional organization of the Drosophila genome.

Nat Commun 2019 10 3;10(1):4485. Epub 2019 Oct 3.

Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.

Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and its functional impact have not been thoroughly investigated. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. We also elucidate the structure of pairing with unprecedented detail, observing significant variation across the genome and revealing at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional implication genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing, including the disruption of some interaction peaks.
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http://dx.doi.org/10.1038/s41467-019-12208-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776532PMC
October 2019

The genome-wide multi-layered architecture of chromosome pairing in early Drosophila embryos.

Nat Commun 2019 10 3;10(1):4486. Epub 2019 Oct 3.

Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA.

Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focus on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first address long-standing questions regarding the structure of embryonic homolog pairing and, to this end, develop a haplotype-resolved Hi-C approach to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which we call Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. We also find a significant genome-wide correlation between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, we demonstrate the versatility of our haplotype-resolved approach by applying it to mammalian embryos.
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http://dx.doi.org/10.1038/s41467-019-12211-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776651PMC
October 2019

Cohesin Members Stag1 and Stag2 Display Distinct Roles in Chromatin Accessibility and Topological Control of HSC Self-Renewal and Differentiation.

Cell Stem Cell 2019 11 5;25(5):682-696.e8. Epub 2019 Sep 5.

Human Oncology and Pathogenesis Program and Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Transcriptional regulators, including the cohesin complex member STAG2, are recurrently mutated in cancer. The role of STAG2 in gene regulation, hematopoiesis, and tumor suppression remains unresolved. We show that Stag2 deletion in hematopoietic stem and progenitor cells (HSPCs) results in altered hematopoietic function, increased self-renewal, and impaired differentiation. Chromatin immunoprecipitation (ChIP) sequencing revealed that, although Stag2 and Stag1 bind a shared set of genomic loci, a component of Stag2 binding sites is unoccupied by Stag1, even in Stag2-deficient HSPCs. Although concurrent loss of Stag2 and Stag1 abrogated hematopoiesis, Stag2 loss alone decreased chromatin accessibility and transcription of lineage-specification genes, including Ebf1 and Pax5, leading to increased self-renewal and reduced HSPC commitment to the B cell lineage. Our data illustrate a role for Stag2 in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation.
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http://dx.doi.org/10.1016/j.stem.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842438PMC
November 2019

Publisher Correction: Heterochromatin drives compartmentalization of inverted and conventional nuclei.

Nature 2019 Aug;572(7771):E22

Institute for Medical Engineering and Science, and Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-019-1454-2DOI Listing
August 2019

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment.

Stem Cell Reports 2019 07 20;13(1):193-206. Epub 2019 Jun 20.

Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306, USA. Electronic address:

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.
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http://dx.doi.org/10.1016/j.stemcr.2019.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627004PMC
July 2019

Heterochromatin drives compartmentalization of inverted and conventional nuclei.

Nature 2019 06 5;570(7761):395-399. Epub 2019 Jun 5.

Institute for Medical Engineering and Science, and Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA.

The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina, preferential attraction of similar chromatin to each other, higher levels of chromatin mobility in active chromatin and transcription-related clustering of euchromatin. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases.
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http://dx.doi.org/10.1038/s41586-019-1275-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206897PMC
June 2019

Measuring the reproducibility and quality of Hi-C data.

Genome Biol 2019 03 19;20(1):57. Epub 2019 Mar 19.

Department of Genome Sciences, University of Washington, Seattle, USA.

Background: Hi-C is currently the most widely used assay to investigate the 3D organization of the genome and to study its role in gene regulation, DNA replication, and disease. However, Hi-C experiments are costly to perform and involve multiple complex experimental steps; thus, accurate methods for measuring the quality and reproducibility of Hi-C data are essential to determine whether the output should be used further in a study.

Results: Using real and simulated data, we profile the performance of several recently proposed methods for assessing reproducibility of population Hi-C data, including HiCRep, GenomeDISCO, HiC-Spector, and QuASAR-Rep. By explicitly controlling noise and sparsity through simulations, we demonstrate the deficiencies of performing simple correlation analysis on pairs of matrices, and we show that methods developed specifically for Hi-C data produce better measures of reproducibility. We also show how to use established measures, such as the ratio of intra- to interchromosomal interactions, and novel ones, such as QuASAR-QC, to identify low-quality experiments.

Conclusions: In this work, we assess reproducibility and quality measures by varying sequencing depth, resolution and noise levels in Hi-C data from 13 cell lines, with two biological replicates each, as well as 176 simulated matrices. Through this extensive validation and benchmarking of Hi-C data, we describe best practices for reproducibility and quality assessment of Hi-C experiments. We make all software publicly available at http://github.com/kundajelab/3DChromatin_ReplicateQC to facilitate adoption in the community.
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http://dx.doi.org/10.1186/s13059-019-1658-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423771PMC
March 2019

Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization.

Cell 2019 03 21;176(6):1502-1515.e10. Epub 2019 Feb 21.

National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

Several general principles of global 3D genome organization have recently been established, including non-random positioning of chromosomes and genes in the cell nucleus, distinct chromatin compartments, and topologically associating domains (TADs). However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are still poorly characterized. Here, we systematically probe heterogeneity in genome organization. High-throughput optical mapping of several hundred intra-chromosomal interactions in individual human fibroblasts demonstrates low association frequencies, which are determined by genomic distance, higher-order chromatin architecture, and chromatin environment. The structure of TADs is variable between individual cells, and inter-TAD associations are common. Furthermore, single-cell analysis reveals independent behavior of individual alleles in single nuclei. Our observations reveal extensive variability and heterogeneity in genome organization at the level of individual alleles and demonstrate the coexistence of a broad spectrum of genome configurations in a cell population.
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http://dx.doi.org/10.1016/j.cell.2019.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408223PMC
March 2019
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