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1.
Cell ; 178(5): 1145-1158.e20, 2019 08 22.
Article in English | MEDLINE | ID: mdl-31402173

ABSTRACT

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.


Subject(s)
Mediator Complex/metabolism , RNA Polymerase II/metabolism , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CRISPR-Cas Systems/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Chromosomal Proteins, Non-Histone/metabolism , Cryoelectron Microscopy , Enhancer Elements, Genetic , Gene Editing , Humans , Male , Mediator Complex/chemistry , Mediator Complex/genetics , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Promoter Regions, Genetic , Protein Structure, Quaternary , RNA Polymerase II/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cohesins
2.
Cell ; 173(5): 1165-1178.e20, 2018 05 17.
Article in English | MEDLINE | ID: mdl-29706548

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Genome , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Line , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomes/metabolism , DNA-Binding Proteins , Humans , Mice , Mutagenesis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Cohesins
3.
Cell ; 170(3): 507-521.e18, 2017 Jul 27.
Article in English | MEDLINE | ID: mdl-28735753

ABSTRACT

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.


Subject(s)
Chromosome Fragility , DNA Breaks, Double-Stranded , Neoplasms/genetics , Animals , B-Lymphocytes/metabolism , CCCTC-Binding Factor , Cell Line, Tumor , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Poly-ADP-Ribose Binding Proteins , Repressor Proteins/metabolism
4.
Cell ; 171(2): 305-320.e24, 2017 Oct 05.
Article in English | MEDLINE | ID: mdl-28985562

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Nucleus/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes/metabolism , Genome, Human , Repressor Proteins/metabolism , CCCTC-Binding Factor , Cell Line, Tumor , DNA-Binding Proteins , Enhancer Elements, Genetic , Histone Code , Humans , Nuclear Proteins/metabolism , Nucleosomes/metabolism , Phosphoproteins/metabolism , Cohesins
5.
Mol Cell ; 84(14): 2648-2664.e10, 2024 Jul 25.
Article in English | MEDLINE | ID: mdl-38955181

ABSTRACT

The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.


Subject(s)
Cryoelectron Microscopy , Cyclin-Dependent Kinase 8 , Mediator Complex , Protein Binding , RNA Polymerase II , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Mediator Complex/metabolism , Mediator Complex/genetics , Mediator Complex/chemistry , Humans , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinase 8/genetics , Animals , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/chemistry , Binding Sites , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , HEK293 Cells , Protein Interaction Domains and Motifs
6.
Mol Cell ; 83(19): 3457-3469.e7, 2023 10 05.
Article in English | MEDLINE | ID: mdl-37802023

ABSTRACT

Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBα, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription in vivo. REV-ERBα condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBα. This work demonstrates physiological circadian protein condensates containing REV-ERBα whose IDR is required for hub formation and the control of rhythmic gene expression.


Subject(s)
Circadian Clocks , Mice , Animals , Circadian Clocks/genetics , Circadian Rhythm/genetics , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Liver/metabolism , Gene Expression
7.
Cell ; 162(4): 708-11, 2015 Aug 13.
Article in English | MEDLINE | ID: mdl-26276627

ABSTRACT

We discuss how principles of nuclear architecture drive typical gene rearrangements in B lymphocytes, whereas translocation hot spots and recurrent lesions reflect the extent of AID-mediated DNA damage and selection.


Subject(s)
B-Lymphocytes/metabolism , Cell Nucleus/metabolism , Gene Rearrangement, B-Lymphocyte , Animals , B-Lymphocytes/cytology , CCCTC-Binding Factor , Cell Nucleus/genetics , Humans , Lymphocyte Activation , Repressor Proteins/metabolism , V(D)J Recombination
8.
Cell ; 162(4): 751-65, 2015 Aug 13.
Article in English | MEDLINE | ID: mdl-26234156

ABSTRACT

The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of "cryptic" recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes.


Subject(s)
Genomic Instability , Homeodomain Proteins/metabolism , Lymphocytes/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Base Sequence , Binding Sites , Cell Line , DNA-Binding Proteins/metabolism , Humans , Lymphocytes/cytology , Mice , Molecular Sequence Data , Translocation, Genetic , V(D)J Recombination
9.
Mol Cell ; 82(18): 3398-3411.e11, 2022 09 15.
Article in English | MEDLINE | ID: mdl-35863348

ABSTRACT

Regulatory elements activate promoters by recruiting transcription factors (TFs) to specific motifs. Notably, TF-DNA interactions often depend on cooperativity with colocalized partners, suggesting an underlying cis-regulatory syntax. To explore TF cooperativity in mammals, we analyze ∼500 mouse and human primary cells by combining an atlas of TF motifs, footprints, ChIP-seq, transcriptomes, and accessibility. We uncover two TF groups that colocalize with most expressed factors, forming stripes in hierarchical clustering maps. The first group includes lineage-determining factors that occupy DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprises ∼30 SP, KLF, EGR, and ZBTB family members that recognize overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking reveal that USFs impart accessibility to colocalized partners and increase their residence time. Mammalian cells have thus evolved a TF superfamily with overlapping DNA binding that facilitate chromatin accessibility.


Subject(s)
Chromatin , Transcription Factors , Animals , Binding Sites , Chromatin/genetics , DNA/genetics , Humans , Mammals/genetics , Mammals/metabolism , Mice , Mice, Knockout , Protein Binding , Transcription Factors/metabolism
10.
Cell ; 159(7): 1538-48, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25483776

ABSTRACT

Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting "convergent" transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.


Subject(s)
Cytidine Deaminase/metabolism , Enhancer Elements, Genetic , Genomic Instability , Transcription, Genetic , Animals , B-Lymphocytes/metabolism , Humans , Immunoglobulin Class Switching , Mice , Transcription Initiation Site
11.
Cell ; 159(7): 1524-37, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25483777

ABSTRACT

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.


Subject(s)
B-Lymphocytes/metabolism , Carcinogenesis , Cytidine Deaminase/genetics , Enhancer Elements, Genetic , Animals , DNA Damage , Humans , Lymphoma/metabolism , Mice
12.
Cell ; 152(3): 620-32, 2013 Jan 31.
Article in English | MEDLINE | ID: mdl-23352430

ABSTRACT

DNA double-strand breaks (DSBs) in B lymphocytes arise stochastically during replication or as a result of targeted DNA damage by activation-induced cytidine deaminase (AID). Here we identify recurrent, early replicating, and AID-independent DNA lesions, termed early replication fragile sites (ERFSs), by genome-wide localization of DNA repair proteins in B cells subjected to replication stress. ERFSs colocalize with highly expressed gene clusters and are enriched for repetitive elements and CpG dinucleotides. Although distinct from late-replicating common fragile sites (CFS), the stability of ERFSs and CFSs is similarly dependent on the replication-stress response kinase ATR. ERFSs break spontaneously during replication, but their fragility is increased by hydroxyurea, ATR inhibition, or deregulated c-Myc expression. Moreover, greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs. In summary, we have identified a source of spontaneous DNA lesions that drives instability at preferred genomic sites.


Subject(s)
Chromosome Fragile Sites , DNA Replication , Eukaryota/genetics , Genomic Instability , Prokaryotic Cells/physiology , Animals , Biomechanical Phenomena , DNA Repair , Humans
13.
Cell ; 153(5): 988-99, 2013 May 23.
Article in English | MEDLINE | ID: mdl-23706737

ABSTRACT

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.


Subject(s)
B-Lymphocytes/metabolism , Gene Expression Regulation , Lymphocyte Activation , Lymphocytes/metabolism , Promoter Regions, Genetic , Animals , B-Lymphocytes/immunology , Cell Line, Tumor , DNA, Single-Stranded/metabolism , Enhancer Elements, Genetic , Genome-Wide Association Study , Humans , Lymphocytes/cytology , Lymphocytes/immunology , Mice , Transcription Factor TFIIH/metabolism , Transcription, Genetic
14.
Cell ; 155(7): 1507-20, 2013 Dec 19.
Article in English | MEDLINE | ID: mdl-24360274

ABSTRACT

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.


Subject(s)
B-Lymphocytes/metabolism , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Promoter Regions, Genetic , Regulon , Animals , Cell Lineage , Cells, Cultured , CpG Islands , DNA Methylation , Genetic Techniques , Mice , Organ Specificity , RNA, Long Noncoding/genetics , Transcription Factors/metabolism , Transcription, Genetic
16.
Nat Immunol ; 16(7): 766-774, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25985233

ABSTRACT

Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.


Subject(s)
B-Lymphocytes/immunology , Clonal Evolution/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cells, B-Lymphoid/immunology , Adolescent , Animals , Antibody Diversity/genetics , Antibody Diversity/immunology , B-Lymphocytes/metabolism , Child , Child, Preschool , Clonal Evolution/genetics , Cytidine Deaminase/genetics , Cytidine Deaminase/immunology , Cytidine Deaminase/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Female , Flow Cytometry , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Homeodomain Proteins/metabolism , Humans , Immunoblotting , Infant , Male , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Microscopy, Fluorescence , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cells, B-Lymphoid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
17.
Cell ; 151(1): 68-79, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-23021216

ABSTRACT

The c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and differentiation at the cellular, tissue, or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Myc's targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature.


Subject(s)
Embryonic Stem Cells/metabolism , Lymphocytes/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcriptional Activation , Animals , B-Lymphocytes/metabolism , DNA-Directed RNA Polymerases/metabolism , Genome , Humans , Mice , Promoter Regions, Genetic , Spleen/cytology
18.
Nature ; 590(7845): 338-343, 2021 02.
Article in English | MEDLINE | ID: mdl-33442057

ABSTRACT

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH-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-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6-9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH 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 VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, 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 VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.


Subject(s)
B-Lymphocytes/metabolism , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Homeodomain Proteins/metabolism , Immunoglobulin Heavy Chains/genetics , Nucleic Acid Conformation , Animals , B-Lymphocytes/cytology , B-Lymphocytes/enzymology , Cell Line , Cells, Cultured , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Down-Regulation , Endonucleases/deficiency , Endonucleases/genetics , G1 Phase Cell Cycle Checkpoints , High-Throughput Nucleotide Sequencing , Homeodomain Proteins/genetics , Mice , Mice, Inbred C57BL , Proteins/genetics , Proteins/metabolism , V(D)J Recombination/genetics
19.
Nature ; 593(7859): 440-444, 2021 05.
Article in English | MEDLINE | ID: mdl-33767446

ABSTRACT

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. 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.


Subject(s)
DNA Breaks, Single-Stranded , DNA Repair , Enhancer Elements, Genetic/genetics , Neurons/metabolism , 5-Methylcytosine/metabolism , Cell Line , DNA/biosynthesis , DNA Replication , Humans , Male , Methylation , Poly(ADP-ribose) Polymerases/metabolism , Sequence Analysis, DNA
20.
Nature ; 595(7866): 278-282, 2021 07.
Article in English | MEDLINE | ID: mdl-34098567

ABSTRACT

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1-3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.


Subject(s)
Antibodies, Neutralizing/immunology , Camelids, New World/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/isolation & purification , CRISPR-Cas Systems , Camelids, New World/genetics , Female , Gene Editing , Humans , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Mutation , Neutralization Tests , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Single-Domain Antibodies/genetics , Single-Domain Antibodies/isolation & purification , Somatic Hypermutation, Immunoglobulin/genetics
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