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

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

Highly structured homolog pairing reflects functional organization of the Drosophila genome

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. Keywords: Computational biology and bioinformatics; Epigenetics; Functional genomics; Molecular biologyNational Institute of General Medical Sciences (U.S.) (Grant R01HD091797)National Institute of General Medical Sciences (U.S.) (Grant R01GM123289)National Institute of General Medical Sciences (U.S.) (Grant DP1GM106412)National Institute of General Medical Sciences (U.S.) (Grant R01 GM114190

Transcription-mediated supercoiling regulates genome folding and loop formation

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.e acknowledge support from European Union’s Horizon 2020 research and innovation program (CellViewer 686637 to J.S., M.L., and M.P.C.); Ministerio de Ciencia e Innovación (grant BFU2017-86760-P [AEI/FEDER, UE] to M.P.C.), and an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya (2017 SGR 689 to M.P.C.); Centro de Excelencia Severo Ochoa (2013–2017 to M.P.C.); CERCA Programme/Generalitat de Catalunya (to M.P.C.); the Spanish Ministry of Science and Innovation to the European Molecular Biology Laboratory (EMBL) partnership (to M.P.C.); the National Natural Science Foundation of China (31971177 to M.P.C.); the Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110103001 to M.P.C.); a Linda Pechenik Montague Investigator Award (to M.L.); a University of Pennsylvania Epigenetics Pilot Award (to M.L.); the Center for Engineering and Mechanobiology (CEMB) a National Science Foundation (NSF) Science and Technology Center Pilot Award (Division of Civil, Mechanical and Manufacturing Innovation [CMMI]: 15-48571 to M.L.); NIH grants R01HD091797 and R01GM123289 (to C.t.W.); the People Program (Marie Skłodowska-Curie Actions) FP7/2007–2013 under an REA grant (608959 to M.V.N.); Juan de la Cierva-Incorporación 2017 (to M.V.N.); Grant for the recruitment of early-stage research staff FI-2020 (Operational Program of Catalonia 2014–2020 CCI 2014ES05SFOP007 of the European Social Fund to L.M.); a “la Caixa” Foundation Fellowship (ID 100010434, LCF/BQ/DR20/11790016) (to L.M.); “La Caixa-Severo Ochoa” pre-doctoral fellowship (to Á.C.-G.); and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and the co-financing of Fondo Social Europeo (2018FI_B_00637 and FSE to R.S.-P.), and a La Caixa international PhD fellowship (to F.S.).Peer reviewe

Transcription-mediated supercoiling regulates genome folding and loop formation

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo

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

Chromosome organization is crucial for genome function. 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. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.This work was supported by funds from Ministerio de Ciencia, Innovación y Universidades of Spain (http://www.ciencia.gob.es/) (IJCI-2015-23352) to IF, Damon Runyon Cancer Research Foundation (https://www.damonrunyon.org/) and Howard Hughes Medical Institute (https://www.hhmi.org/) to BJB, Uehara Memorial Foundation Research (https://www.taisho-holdings.co.jp/en/environment/social/sciences/) to HMS, William Randolph Hearst Foundation (https://www.hearstfdn.org/) to RBM, EMBO (Long-Term fellowship) (https://www.embo.org/) to JE, NSF (Center for Theoretical Biological Physics, Rice University) (https://www.nsf.gov/) to MDP and JNO, NSF (CCF-1054898, CCF-1317291) (https://www.nsf.gov/), NIH (1R01EB018659-01, 1-U01- MH106011-01) (https://www.nih.gov/), and Office of Naval Research (N00014-13-1-0593, N00014-14-1-0610, N00014-16-1-2182, N00014-16-1- 2410) (https://www.onr.navy.mil/) to PY, NIH (1DP2OD008540, U01HL130010, UM1HG009375, 4DP2OD008540) (https://www.nih.gov/), NSF (PHY-1427654) (https://www.nsf.gov/), USDA (2017-05741) (https://www.usda.gov/), Welch Foundation (Q-1866) (http://www.welch1.org/), NVIDIA (https://www.nvidia.com/en-us/), IBM (https://www.ibm.com/us-en/?lnk=m), Google (https://www.google.com/), Cancer Prevention Research Institute of Texas (R1304) (http://www.cprit.state.tx.us/), and McNair Medical Institute (http://www.mcnairfoundation.org/what-we-fund/mcnair-medical-institute/) to E.L.A., Horizon 2020 Research and Innovation Programme (676556) (https://ec.europa.eu/programmes/horizon2020/en/), European Research Council (609989) (https://erc.europa.eu/), Ministerio de Ciencia, Innovación y Universidades of Spain (BFU2017-85926-P) (http://www.ciencia.gob.es/), CERCA, and AGAUR Programme of the Generalitat de Catalunya and Centros de Excelencia Severo Ochoa (SEV-2012-0208) (http://www.ciencia.gob.es/portal/site/MICINN/menuitem.7eeac5cd345b4f34f09dfd1001432ea0/?vgnextoid=cba733a6368c2310VgnVCM1000001d04140aRCRD) to M.A.M-R., and NIH (5DP1GM106412, R01HD091797, R01GM123289) (https://www.nih.gov/) to C-tW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript