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 , CohesinsABSTRACT
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 , CohesinsABSTRACT
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/metabolismABSTRACT
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 , CohesinsABSTRACT
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 RecombinationABSTRACT
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 â¼500 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 â¼30 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/metabolismABSTRACT
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 SiteABSTRACT
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 , MiceABSTRACT
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, â¼90% 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, GeneticABSTRACT
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, GeneticABSTRACT
The RAG endonuclease initiates Igh locus V(D)J recombination in progenitor (pro)-B cells1. Upon binding a recombination centre-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJH-based recombination centre2. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG scanning3-5; however, their inactivation allows scanning to proximal VHs, where additional CBEs activate rearrangement and impede scanning any further upstream5. Distal VH utilization is thought to involve diffusional access to the recombination centre following large-scale Igh locus contraction6-8. Here we test the potential of linear RAG scanning to mediate distal VH usage in G1-arrested v-Abl pro-B cell lines9, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably owing to lack of locus contraction2,5. Through an auxin-inducible approach10, we degraded the cohesin component RAD2110-12 or CTCF12,13 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-JH joining, in which synapsis occurs by diffusion2. Remarkably, while degradation of CTCF suppressed most CBE-based chromatin interactions, it promoted robust recombination centre interactions with, and robust VH-to-DJH joining of, distal VHs, 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.
Subject(s)
CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , V(D)J Recombination , Animals , Cell Line , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Female , G1 Phase , Genes, Immunoglobulin Heavy Chain/genetics , Humans , Indoleacetic Acids/metabolism , Male , Mice , Mice, Inbred C57BL , Precursor Cells, B-Lymphoid/immunology , Precursor Cells, B-Lymphoid/metabolism , Transcription, Genetic , V(D)J Recombination/genetics , CohesinsABSTRACT
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.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Immunoglobulin Class Switching/genetics , Immunoglobulin Heavy Chains/genetics , Tumor Suppressor Proteins/genetics , Animals , B-Lymphocytes , Cell Line , Chromatin/genetics , Chromatin/metabolism , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA/genetics , Enhancer Elements, Genetic , Gene Rearrangement , Humans , MYND Domains , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Somatic Hypermutation, Immunoglobulin/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
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.
Subject(s)
B-Lymphocytes/metabolism , Cell Cycle , Cell Nucleus/metabolism , Chromatin Assembly and Disassembly , Chromatin/metabolism , Histones/metabolism , Lymphocyte Activation , Proto-Oncogene Proteins c-myc/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Animals , B-Lymphocytes/immunology , Cell Line , Chromatin/chemistry , Chromatin/genetics , DNA Methylation , Epigenesis, Genetic , Genotype , Histones/chemistry , Immunity, Humoral , Methylation , Mice, Inbred C57BL , Mice, Knockout , Nucleic Acid Conformation , Phenotype , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/genetics , Single Molecule Imaging , Structure-Activity Relationship , Time Factors , Transcription, GeneticABSTRACT
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.
Subject(s)
DNA/metabolism , Genomics/methods , In Situ Hybridization, Fluorescence/methods , Microscopy/methods , Chromosome Painting , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Image Processing, Computer-Assisted , Sequence Analysis, DNA/methodsABSTRACT
Deficiencies in factors that regulate the DNA damage response enhance the incidence of malignancy by destabilizing the genome. However, the precise influence of the DNA damage response on regulation of cancer-associated rearrangements is not well defined. Here we examine the genome-wide impact of tumor protein P53-binding protein 1 (53BP1) deficiency in lymphoma and translocation. While both activation-induced cytidine deaminase (AID) and 53BP1 have been associated with cancer in humans, neither AID overexpression nor loss of 53BP1 is sufficient to produce malignancy. However, the combination of 53BP1 deficiency and AID deregulation results in B cell lymphoma. Deep sequencing of the genome of 53BP1(-/-) cancer cells and translocation capture sequencing (TC-Seq) of primary 53BP1(-/-) B cells revealed that their chromosomal rearrangements differ from those found in wild-type cells in that they show increased DNA end resection. Moreover, loss of 53BP1 alters the translocatome by increasing rearrangements to intergenic regions.
Subject(s)
Cell Transformation, Neoplastic/genetics , Chromosomal Proteins, Non-Histone/physiology , Cytidine Deaminase/physiology , DNA-Binding Proteins/physiology , Gene Rearrangement , Lymphoma, B-Cell/metabolism , Animals , Cells, Cultured , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Mammalian/genetics , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Genes, Tumor Suppressor , Genome-Wide Association Study , Lymphoma, B-Cell/genetics , Mice , Mice, Knockout , Mutation , Sequence Analysis, DNA , Transcription, Genetic , Translocation, Genetic , Tumor Suppressor p53-Binding Protein 1ABSTRACT
Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.
Subject(s)
B-Lymphocytes/metabolism , B-Lymphocytes/pathology , DNA Damage/genetics , Translocation, Genetic/genetics , Animals , B-Lymphocytes/cytology , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Chromosome Positioning , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , Cytidine Deaminase/deficiency , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA Breaks, Double-Stranded , Genes, myc/genetics , Genome/genetics , Immunoglobulin Heavy Chains/genetics , Mice , Replication Protein A/metabolismABSTRACT
Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by cutaneous fibrofolliculomas, pulmonary cysts, and kidney malignancies. Affected individuals carry germ line mutations in folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney cancer, FLCN has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germ line deletion of Flcn results in early embryonic lethality in animal models. Here, we describe mice deficient in the newly characterized folliculin-interacting protein 1 (Fnip1). In contrast to Flcn, Fnip1(-/-) mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is entirely independent of mTOR activity. We show that this developmental arrest results from rapid caspase-induced pre-B cell death, and that a Bcl2 transgene reconstitutes mature B-cell populations, respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1(-/-) mice. Our studies thus demonstrate that the FLCN-FNIP complex deregulated in BHD syndrome is absolutely required for B-cell differentiation, and that it functions through both mTOR-dependent and independent pathways.
Subject(s)
B-Lymphocytes/physiology , Birt-Hogg-Dube Syndrome/genetics , Carrier Proteins/genetics , Cell Differentiation/genetics , Gene Deletion , Proto-Oncogene Proteins/genetics , Tumor Suppressor Proteins/genetics , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Carrier Proteins/metabolism , Carrier Proteins/physiology , Cell Differentiation/immunology , Cells, Cultured , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Signal Transduction/genetics , Signal Transduction/physiology , Species Specificity , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/physiologyABSTRACT
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.
Subject(s)
DNA Footprinting/methods , DNA/chemistry , DNA/ultrastructure , Animals , Chromatin , Computational Biology/methods , DNA, Single-Stranded/ultrastructure , Fungal Proteins , G-Quadruplexes , Gene Expression Regulation/physiology , Genome , Mammals/genetics , Manganese Compounds/metabolism , Mice , Nucleic Acid Conformation , Nucleosomes , Oxides/metabolism , Protein Binding , Single-Strand Specific DNA and RNA EndonucleasesABSTRACT
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.