Publications by authors named "Rafael Casellas"

79 Publications

Neuronal enhancers are hotspots for DNA single-strand break repair.

Nature 2021 Mar 25. Epub 2021 Mar 25.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
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http://dx.doi.org/10.1038/s41586-021-03468-5DOI Listing
March 2021

Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants.

bioRxiv 2021 Mar 4. Epub 2021 Mar 4.

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.
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http://dx.doi.org/10.1101/2021.03.04.433768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941635PMC
March 2021

Structure of mammalian Mediator complex reveals Tail module architecture and interaction with a conserved core.

Nat Commun 2021 03 1;12(1):1355. Epub 2021 Mar 1.

Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical School, Aurora, CO, USA.

The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals that the previously unresolved Tail module, which includes a number of metazoan specific subunits, interacts extensively with core Mediator and has the potential to influence its conformation and interactions.
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http://dx.doi.org/10.1038/s41467-021-21601-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921410PMC
March 2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Nature 2021 Feb 10. Epub 2021 Feb 10.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA.

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.
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http://dx.doi.org/10.1038/s41586-021-03324-6DOI Listing
February 2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected nearly 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease-2019 (COVID-19) including two novel mRNA-based vaccines . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known . Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S), receptor binding domain (RBD) binding titers . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection . However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.
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http://dx.doi.org/10.1101/2021.01.15.426911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836122PMC
January 2021

Loop extrusion mediates physiological Igh locus contraction for RAG scanning.

Nature 2021 02 13;590(7845):338-343. Epub 2021 Jan 13.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning. Scanning of RAG from the DJ-RC-RSS to upstream convergent V-RSSs is impeded by D-proximal CTCF-binding elements (CBEs). Primary progenitor B cells undergo a mechanistically undefined contraction of the V locus that is proposed to provide distal Vs access to the DJ-RC. Here we report that an inversion of the entire 2.4-Mb V locus in mouse primary progenitor B cells abrogates rearrangement of both V-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic V-RSSs that are normally in opposite orientation and RAG scanning beyond the V locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the V locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL), a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the V locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.
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http://dx.doi.org/10.1038/s41586-020-03121-7DOI Listing
February 2021

Genome-wide mutational signatures revealed distinct developmental paths for human B cell lymphomas.

J Exp Med 2021 Feb;218(2)

Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China.

Both somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase (AID). Dysregulation of these processes has been linked to B cell lymphomagenesis. Here we performed an in-depth analysis of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) genomes. We characterized seven genomic mutational signatures, including two B cell tumor-specific signatures, one of which is novel and associated with aberrant SHM. We further identified two major mutational signatures (K1 and K2) of clustered mutations (kataegis) resulting from the activities of AID or error-prone DNA polymerase η, respectively. K1 was associated with the immunoglobulin (Ig) switch region mutations/translocations and the ABC subtype of DLBCL, whereas K2 was related to the Ig variable region mutations and the GCB subtype of DLBCL and FL. Similar patterns were also observed in chronic lymphocytic leukemia subtypes. Thus, alterations associated with aberrant CSR and SHM activities can be linked to distinct developmental paths for different subtypes of B cell lymphomas.
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http://dx.doi.org/10.1084/jem.20200573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608067PMC
February 2021

CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning.

Nature 2020 10 27;586(7828):305-310. Epub 2020 Jul 27.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.

The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells. Upon binding a recombination centre-based J, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ-based recombination centre. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning; however, their inactivation allows scanning to proximal Vs, where additional CBEs activate rearrangement and impede scanning any further upstream. Distal V utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction. Here we test the potential of linear RAG scanning to mediate distal V usage in G1-arrested v-Abl pro-B cell lines, which undergo robust D-to-J but little V-to-DJ rearrangements, presumably owing to lack of locus contraction. Through an auxin-inducible approach, we degraded the cohesin component RAD21 or CTCF in these G1-arrested lines. Degradation of RAD21 eliminated all V(D)J recombination and interactions associated with RAG scanning, except for reecombination centre-located DQ52-to-J joining, in which synapsis occurs by diffusion. Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust V-to-DJ joining of, distal Vs, with patterns similar to those of 'locus-contracted' primary pro-B cells. Thus, downmodulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG scanning across the 2.7-Mb Igh locus.
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http://dx.doi.org/10.1038/s41586-020-2578-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554077PMC
October 2020

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells.

Cell Res 2020 09 30;30(9):732-744. Epub 2020 Apr 30.

State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.
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http://dx.doi.org/10.1038/s41422-020-0328-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608219PMC
September 2020

3D ATAC-PALM: super-resolution imaging of the accessible genome.

Nat Methods 2020 04 16;17(4):430-436. Epub 2020 Mar 16.

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.
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http://dx.doi.org/10.1038/s41592-020-0775-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7207063PMC
April 2020

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation.

Cell Rep 2019 12;29(12):3902-3915.e8

Department of Immunobiology, Yale School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011, USA. Electronic address:

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible ("hot") and resistant ("cold") to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.
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http://dx.doi.org/10.1016/j.celrep.2019.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980758PMC
December 2019

Fundamental roles of chromatin loop extrusion in antibody class switching.

Nature 2019 11 30;575(7782):385-389. Epub 2019 Oct 30.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμ constant region exons (Cs) with one of six Cs lying 100-200 kb downstream. Each C is flanked upstream by an I promoter and long repetitive switch (S) region. Cytokines and activators induce activation-induced cytidine deaminase (AID) and I-promoter transcription, with 3' IgH regulatory region (3' IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3' IgHRR interactions. Transcription through donor Sμ and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an Sμ-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining. Here we report that chromatin loop extrusion underlies the mechanism by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3' IgHRR enhancers with the 200-kb upstream Sμ to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with Sμ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with Sμ in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.
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http://dx.doi.org/10.1038/s41586-019-1723-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856444PMC
November 2019

In Vivo Chemical Probing for G-Quadruplex Formation.

Methods Mol Biol 2019 ;2035:369-382

Laboratory of Pathology, National Cancer Institute, National Institutes of Health (USA), Bethesda, MD, USA.

While DNA inside the cells is predominantly canonical right-handed double helix, guanine-rich DNAs have potential to fold into four-stranded structures that contain stacks of G-quartets (G4 DNA quadruplex). Genome sequencing has revealed G4 sequences tend to localize at the gene control regions, especially in the promoters of oncogenes. A growing body of evidence indicates that G4 DNA quadruplexes might have important regulatory roles in genome function, highlighting the need for techniques to detect genome-wide folding of DNA into this structure. Potassium permanganate in vivo treatment of cells results in oxidizing of nucleotides in single-stranded DNA regions that accompany G4 DNA quadruplexes formation, providing an excellent probe for the conformational state of DNA inside the living cells. Here, we describe a permanganate-based methodology to detect G4 DNA quadruplex, genome-wide. This methodology combined with high-throughput sequencing provides a snapshot of the DNA conformation over the whole genome in vivo.
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http://dx.doi.org/10.1007/978-1-4939-9666-7_23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444663PMC
April 2020

A Pliable Mediator Acts as a Functional Rather Than an Architectural Bridge between Promoters and Enhancers.

Cell 2019 08 8;178(5):1145-1158.e20. Epub 2019 Aug 8.

Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD 20892, USA; Center for Cancer Research, NCI, NIH, Bethesda, MD 20892, USA. Electronic address:

While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
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http://dx.doi.org/10.1016/j.cell.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533040PMC
August 2019

The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1.

Mol Cell 2019 09 31;75(6):1229-1242.e5. Epub 2019 Jul 31.

Lymphocyte Cell Biology Section, Molecular Immunology and Inflammation Branch, NIAMS, NIH, Bethesda, MD 20892, USA. Electronic address:

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.
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http://dx.doi.org/10.1016/j.molcel.2019.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754279PMC
September 2019

Gain of CTCF-Anchored Chromatin Loops Marks the Exit from Naive Pluripotency.

Cell Syst 2018 11 7;7(5):482-495.e10. Epub 2018 Nov 7.

European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, Heidelberg 69117, Germany. Electronic address:

The genome of pluripotent stem cells adopts a unique three-dimensional architecture featuring weakly condensed heterochromatin and large nucleosome-free regions. Yet, it is unknown whether structural loops and contact domains display characteristics that distinguish embryonic stem cells (ESCs) from differentiated cell types. We used genome-wide chromosome conformation capture and super-resolution imaging to determine nuclear organization in mouse ESC and neural stem cell (NSC) derivatives. We found that loss of pluripotency is accompanied by widespread gain of structural loops. This general architectural change correlates with enhanced binding of CTCF and cohesins and more pronounced insulation of contacts across chromatin boundaries in lineage-committed cells. Reprogramming NSCs to pluripotency restores the unique features of ESC domain topology. Domains defined by the anchors of loops established upon differentiation are enriched for developmental genes. Chromatin loop formation is a pervasive structural alteration to the genome that accompanies exit from pluripotency and delineates the spatial segregation of developmentally regulated genes.
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http://dx.doi.org/10.1016/j.cels.2018.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327227PMC
November 2018

Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

Mol Cell 2018 10 4;72(2):222-238.e11. Epub 2018 Oct 4.

Abramson Family Cancer Research Institute and Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.
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http://dx.doi.org/10.1016/j.molcel.2018.08.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407864PMC
October 2018

The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination.

Mol Cell 2018 11 4;72(4):636-649.e8. Epub 2018 Oct 4.

Laboratory of DNA Repair and Maintenance of Genome Stability, The Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin 13125, Germany. Electronic address:

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.
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http://dx.doi.org/10.1016/j.molcel.2018.08.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242708PMC
November 2018

The Energetics and Physiological Impact of Cohesin Extrusion.

Cell 2018 05 26;173(5):1165-1178.e20. Epub 2018 Apr 26.

Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD 20892, USA; Center of Cancer Research, NCI, NIH, Bethesda, MD 20892, USA. Electronic address:

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.
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http://dx.doi.org/10.1016/j.cell.2018.03.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065110PMC
May 2018

Epigenetic silencing of miR-125b is required for normal B-cell development.

Blood 2018 04 19;131(17):1920-1930. Epub 2018 Mar 19.

Division of Biology and Biological Engineering and.

Deregulation of several microRNAs (miRs) can influence critical developmental checkpoints during hematopoiesis as well as cell functions, eventually leading to the development of autoimmune disease or cancer. We found that miR-125b is expressed in bone marrow multipotent progenitors and myeloid cells but shut down in the B-cell lineage, and the gene encoding miR-125b lacked transcriptional activation markers in B cells. To understand the biological importance of the physiological silencing of miR-125b expression in B cells, we drove its expression in the B-cell lineage and found that dysregulated miR-125b expression impaired egress of immature B cells from the bone marrow to peripheral blood. Such impairment appeared to be mediated primarily by inhibited expression of the sphingosine-1-phosphate receptor 1 (S1PR1). Enforced expression of S1PR1 or clustered regularly interspaced short palindromic repeats/Cas9-mediated genome editing of the miR-125b targeting site in the S1PR1 3' untranslated region rescued the miR-125b-mediated defect in B-cell egress. In addition to impaired B-cell egress, miR-125b dysregulation initially reduced pre-B-cell output but later induced pre-B-cell lymphoma/leukemia in mice. Genetic deletion of IRF4 was found in miR-125b-induced B-cell cancer, but its role in oncogenic miR-125b-induced B-cell transformation is still unknown. Here, we further demonstrated an interaction of the effects of miR-125b and IRF4 in cancer induction by showing that miR125b-induced B-cell leukemia was greatly accelerated in IRF4 homozygous mutant mice. Thus, we conclude that physiological silencing of miR-125b is required for normal B-cell development and also acts as a mechanism of cancer suppression.
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http://dx.doi.org/10.1182/blood-2018-01-824540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5921965PMC
April 2018

The Reign of Antibodies: A Celebration of and Tribute to Michael Potter and His Homogeneous Immunoglobulin Workshops.

J Immunol 2018 01;200(1):23-26

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104

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http://dx.doi.org/10.4049/jimmunol.1701516DOI Listing
January 2018

Cohesin Loss Eliminates All Loop Domains.

Cell 2017 Oct;171(2):305-320.e24

The Center for Genome Architecture, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Center for Theoretical Biological Physics, Rice University, Houston, TX 77030, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA; Departments of Computer Science and Computational and Applied Mathematics, Rice University, Houston, TX 77030, USA. Electronic address:

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. 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. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.
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http://dx.doi.org/10.1016/j.cell.2017.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846482PMC
October 2017

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

Mol Cell 2017 Aug 10;67(4):566-578.e10. Epub 2017 Aug 10.

Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD 20892, USA; Center of Cancer Research, NCI, NIH, Bethesda, MD 20892, USA. Electronic address:

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
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http://dx.doi.org/10.1016/j.molcel.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854204PMC
August 2017

Genome Organization Drives Chromosome Fragility.

Cell 2017 Jul 20;170(3):507-521.e18. Epub 2017 Jul 20.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2017.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133249PMC
July 2017

Permanganate/S1 Nuclease Footprinting Reveals Non-B DNA Structures with Regulatory Potential across a Mammalian Genome.

Cell Syst 2017 03 22;4(3):344-356.e7. Epub 2017 Feb 22.

Laboratory of Pathology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

DNA in cells is predominantly B-form double helix. Though certain DNA sequences in vitro may fold into other structures, such as triplex, left-handed Z form, or quadruplex DNA, the stability and prevalence of these structures in vivo are not known. Here, using computational analysis of sequence motifs, RNA polymerase II binding data, and genome-wide potassium permanganate-dependent nuclease footprinting data, we map thousands of putative non-B DNA sites at high resolution in mouse B cells. Computational analysis associates these non-B DNAs with particular structures and indicates that they form at locations compatible with an involvement in gene regulation. Further analyses support the notion that non-B DNA structure formation influences the occupancy and positioning of nucleosomes in chromatin. These results suggest that non-B DNAs contribute to the control of a variety of critical cellular and organismal processes.
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http://dx.doi.org/10.1016/j.cels.2017.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432990PMC
March 2017

Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction.

Nat Commun 2016 12 23;7:13895. Epub 2016 Dec 23.

Immunoregulation Section, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), Intramural Research Program, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA.

Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.
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http://dx.doi.org/10.1038/ncomms13895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5196435PMC
December 2016

The cell cycle restricts activation-induced cytidine deaminase activity to early G1.

J Exp Med 2017 01 20;214(1):49-58. Epub 2016 Dec 20.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065

Activation-induced cytidine deaminase (AID) converts cytosine into uracil to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes. In addition, this enzyme produces DNA lesions at off-target sites that lead to mutations and chromosome translocations. However, AID is mostly cytoplasmic, and how and exactly when it accesses nuclear DNA remains enigmatic. Here, we show that AID is transiently in spatial contact with genomic DNA from the time the nuclear membrane breaks down in prometaphase until early G1, when it is actively exported into the cytoplasm. Consistent with this observation, the immunoglobulin (Igh) gene deamination as measured by uracil accumulation occurs primarily in early G1 after chromosomes decondense. Altering the timing of cell cycle-regulated AID nuclear residence increases DNA damage at off-target sites. Thus, the cell cycle-controlled breakdown and reassembly of the nuclear membrane and the restoration of transcription after mitosis constitute an essential time window for AID-induced deamination, and provide a novel DNA damage mechanism restricted to early G1.
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http://dx.doi.org/10.1084/jem.20161649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206505PMC
January 2017

Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells.

Cell Rep 2016 10;17(5):1369-1382

Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.
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http://dx.doi.org/10.1016/j.celrep.2016.09.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123747PMC
October 2016