Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals.

Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling. [Abstract copyright: Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Retromer Regulates Postendocytic Sorting of β-Secretase in Polarized Madin–Darby Canine Kidney Cells

β-Amyloid (Aβ) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the β-APP cleaving enzyme (BACE or β-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin–Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aβ peptides.This research was supported by a grant from the Spanish Ministerio de Sanidad y Consumo to M. Vergés (PI07/0895). Y. Cuartero was a recipient of a predoctoral fellowship from the CIPF (PR 01/2007), and M. Verges, of a Ramón y Cajal contract from the Spanish Ministerio de Educación y Ciencia. A. Capell was supported by the Helmholtz Alliance for Mental Health in an Ageing Society.Peer reviewe

OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes

The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb

Parallel sequencing lives, or what makes large sequencing projects successful

T47D rep2 and b1913e6c1 51720e9cf were 2 Hi-C samples. They were born and processed at the same time, yet their fates were very different. The life of b1913e6c1 51720e9cf was simple and fruitful, while that of T47D rep2 was full of accidents and sorrow. At the heart of these differences lies the fact that b1913e6c1 51720e9cf was born under a lab culture of Documentation, Automation, Traceability, and Autonomy and compliance with the FAIR Principles. Their lives are a lesson for those who wish to embark on the journey of managing high-throughput sequencing data

Rapid reversible changes in compartments and local chromatin organization revealed by hyperosmotic shock

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.The study was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64437-P and FEDER, BFU2014-52125-REDT, and BFU2014-51672-REDC to F.P.; BFU2017-85152-P and FEDER to E.d.N.), the Catalan Government (2017 SGR 799), the Fundación Botín, and the Banco Santander through its Santander Universities Global Division to F.P. and the Unidad de Excelencia Maria de Maeztu, MDM-2014-0370. F.P. is recipient of an ICREA Acadèmia (Generalitat de Catalunya). M.B. received the support of the European Research Council (ERC Synergy Grant 4D Genome 609989) and CERCA

Specific contributions of cohesin-SA1 and cohesin-SA2 to TADs and polycomb domains in embryonic stem cells

Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.This work was funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (FEDER) (grant BFU2016-79841-R to A.L.), Comunidad de Madrid (contract PEJD-2016/BMD-3190 to G.M.-S.), Centro de Excelencia Severo Ochoa to CNIO (SEV-2015-0510), and the National Institute of Health Carlos III (ISCIII). The work of Y.C. and M.A.M.-R. was partially supported by the European Research Council (ERC) under the Seventh Framework Programme FP7/2007–2013 (ERC grant agreement 609989) and the European Union’s Horizon 2020 research and innovation program (grant agreement 676556). M.A.M.-R. also acknowledges support from the Spanish Ministry of Economy and Competitiveness (BFU2017-85926-P and the Centro de Excelencia Severo Ochoa to Center for Genomic Regulation), and the Generalitat de Catalunya (AGAUR grant SGR468 and CERCA Programme)

OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes

The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb.Spanish Ministry of Economy and Competitiveness ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]; ACER (to C.R.G.); EMBO Long-term Fellowship [ALTF 1201-2014 to R.S.]; Marie Curie Individual Fellowship [H2020-MSCA-IF-2014]; European Research Council under the European Union’s Seventh Framework Programme [FP7/2007–2013)/ERC Synergy grant agreement 609989 (4DGenome)]. We acknowledge the support of the CERCA Programme / Generalitat de Catalunya. Funding for open access charge: European Research Council

Specific contributions of cohesin-SA1 and cohesin-SA2 to TADs and polycomb domains in embryonic stem cells

Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.This work was funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (FEDER) (grant BFU2016-79841-R to A.L.), Comunidad de Madrid (contract PEJD-2016/BMD-3190 to G.M.-S.), Centro de Excelencia Severo Ochoa to CNIO (SEV-2015-0510), and the National Institute of Health Carlos III (ISCIII). The work of Y.C. and M.A.M.-R. was partially supported by the European Research Council (ERC) under the Seventh Framework Programme FP7/2007–2013 (ERC grant agreement 609989) and the European Union’s Horizon 2020 research and innovation program (grant agreement 676556). M.A.M.-R. also acknowledges support from the Spanish Ministry of Economy and Competitiveness (BFU2017-85926-P and the Centro de Excelencia Severo Ochoa to Center for Genomic Regulation), and the Generalitat de Catalunya (AGAUR grant SGR468 and CERCA Programme)

Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals

Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling.This work was supported by the Ministry of Economy, Industry and Competitiveness (CGL2017-83802-P to A.R.-H.) and the Spanish Ministry of Science and Innovation (PID2020-112557GB-I00 to A.R.-H. and PID2020-115696RB-I00 to M.A.M.-R.). Research funding to P.D.W. (Australian Research Council grants DP180100931, DP210103512, and DP220101429) and T.J.R. (South African National Research Foundation) are gratefully acknowledged. C.V. and L.A.-G. were supported by FPI predoctoral fellowships from the Ministry of Economy and Competitiveness (BES-2015-072924 and PRE-2018-083257). L.M.-G. was supported by an FPU predoctoral fellowship from the Spanish Ministry of Science, Innovation and University (FPU18/03867). C.A.-S. was supported by a GTA fellowship from the University of Kent

STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.Fundación Científica de la Asociación Española Contra el Cáncer (to F.X.R., E.L., in part); V.P. is supported by INSERM, the Fondation Toulouse Cancer Santé and Pierre Fabre Research Institute as part of the Chair of Bioinformatics in Oncology of the CRCT; Bioinfo4women programme at the Barcelona Supercomputing Center; European Union's H2020 Framework Programme through the ERC [609989 to M.A.M.-R., in part]; Spanish Ministerio de Ciencia, Innovación y Universidades [BFU2017-85926-P to M.A.M.-R.]; C.N.I.O. is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa [SEV-2015-0510]; C.R.G. acknowledges support from ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]; Spanish ministry of Science and Innovation to the EMBL partnership and the CERCA Programme/Generalitat de Catalunya (to C.R.G.); C.R.G. also acknowledges support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement; Spanish Ministry of Science and Innovation with funds from the European Regional Development Fund (ERDF) corresponding to the 2014–2020 Smart Growth Operating Program (to C.N.A.G.). Funding for open access charge: Own funds