RAG Represents a Widespread Threat to the Lymphocyte Genome.

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.

Histone reader BRWD1 targets and restricts recombination to the Igk locus.

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

Increasing the calcium sensitivity of muscle using trifluoperazine-induced manipulations in silico, in vitro and in vivo systems.

Many therapeutics for cardiomyopathy treat the symptoms of the disease rather than the underlying mechanism. The mechanism of cardiomyopathy onset is believed to include two means: calcium sensitivity changes and myosin activity alteration. Trifluoperazine is a compound that binds troponin, and other components of the calcium pathway, which impacts calcium regulation of contraction. Here, the ability of TFP to shift calcium sensitivity was examined in vitro with purified proteins and the impact of TFP on heart function was assessed in vivo using embryonic zebrafish. The binding of TFP to troponin was modeled in silico and a model of zebrafish troponin was generated. TFP increased regulated cardiac actomyosin activity in vitro and elevated embryonic zebrafish heart rates at effective drug concentrations. Troponin structural changes predicted in silico suggest altered protein interactions within thin filaments that would affect the regulation of heart function.

The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development.

Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are the result of changes in protein interactions between ACTC protein and force-generating ß-myosin or the calcium-dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin relative to the myosin or regulatory protein binding sites on what we call the "dark side" of actin. The T126I change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomyopathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.

M-class hypertrophic cardiomyopathy cardiac actin mutations increase calcium sensitivity of regulated thin filaments.

Hypertrophic cardiomyopathy is a commonly occurring cardiovascular disease resulting primarily from changes in proteins participating in muscle contraction in the heart, including the cardiac actin protein. Changes in cardiac actin located exclusively in the myosin binding site are called M-class variants and include the H88Y, R95C, and E99K substitutions and F90Δ deletion. The prevailing hypothesis for these mutations is that hypertrophic cardiomyopathy is the result of increased calcium sensitivity of contraction in the myocardium. To test this hypothesis, we studied the activity of myosin at varying calcium concentrations in the presence of regulated thin filaments containing M-class cardiac actin variants. We found that all M-class cardiac actin variants exhibit increased calcium sensitivity, with the R95C variant also displaying significant decreases in maximal myosin activity. This work represents the first characterization of all M-class variant proteins and suggests that drugs targeting contraction specifically to treat hypertrophic cardiomyopathy must consider the impact on both calcium sensitivity and maximal myosin activity on overall heart health.

Clonal allelic predetermination of immunoglobulin-κ rearrangement.

Although most genes are expressed biallelically, a number of key genomic sites--including immune and olfactory receptor regions--are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.

RAG1 targeting in the genome is dominated by chromatin interactions mediated by the non-core regions of RAG1 and RAG2.

The RAG1/RAG2 endonuclease initiates V(D)J recombination at antigen receptor loci but also binds to thousands of places outside of these loci. RAG2 localizes directly to lysine 4 trimethylated histone 3 (H3K4me3) through a plant homeodomain (PHD) finger. The relative contribution of RAG2-dependent and RAG1-intrinsic mechanisms in determining RAG1 binding patterns is not known. Through analysis of deep RAG1 ChIP-seq data, we provide a quantitative description of the forces underlying genome-wide targeting of RAG1. Surprisingly, sequence-specific DNA binding contributes minimally to RAG1 targeting outside of antigen receptor loci. Instead, RAG1 binding is driven by two distinct modes of interaction with chromatin: the first is driven by H3K4me3, promoter-focused and dependent on the RAG2 PHD, and the second is defined by H3K27Ac, enhancer-focused and dependent on 'non-core' portions of RAG1. Based on this and additional chromatin and genomic features, we formulated a predictive model of RAG1 targeting to the genome. RAG1 binding sites predicted by our model correlate well with observed patterns of RAG1-mediated breaks in human pro-B acute lymphoblastic leukemia. Overall, this study provides an integrative model for RAG1 genome-wide binding and off-target activity and reveals a novel role for the RAG1 non-core region in RAG1 targeting.

MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase.

B lymphocytes perform somatic hypermutation and class-switch recombination (CSR) of the immunoglobulin locus to generate an antibody repertoire diverse in both affinity and function. These somatic diversification processes are catalyzed by activation-induced cytidine deaminase (AID), a potent DNA mutator whose expression and function are highly regulated. Here we show that AID was regulated posttranscriptionally by a lymphocyte-specific microRNA, miR-155. We found that miR-155 was upregulated in murine B lymphocytes undergoing CSR and that it targeted a conserved site in the 3'-untranslated region of the mRNA encoding AID. Disruption of this target site in vivo resulted in quantitative and temporal deregulation of AID expression, along with functional consequences for CSR and affinity maturation. Thus, miR-155, which has recently been shown to play important roles in regulating the germinal-center reaction, does so in part by directly downmodulating AID expression.

A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation.

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.

Peripheral subnuclear positioning suppresses Tcrb recombination and segregates Tcrb alleles from RAG2.

Allelic exclusion requires that the two alleles at antigen-receptor loci attempt to recombine variable (V), diversity (D), and joining (J) gene segments [V(D)J recombination] asynchronously in nuclei of developing lymphocytes. It previously was shown that T-cell receptor ß (Tcrb) alleles frequently and stochastically associate with the nuclear lamina and pericentromeric heterochromatin in CD4(-)CD8(-) thymocytes. Moreover, rearranged alleles were underrepresented at these locations. Here we used 3D immunofluorescence in situ hybridization to identify recently rearranged Tcrb alleles based on the accumulation of the DNA-repair protein 53BP1. We found that Tcrb alleles recombine asynchronously in double-negative thymocytes and that V(D)J recombination is suppressed on peripheral as compared with central Tcrb alleles. Moreover, the recombination events that did take place at the nuclear periphery preferentially occurred on Tcrb alleles that were partially dissociated from the nuclear lamina. To understand better the mechanism by which V(D)J recombination is suppressed at the nuclear periphery, we evaluated the subnuclear distribution of recombination-activating gene 2 (RAG2) protein. We found that RAG2 abundance was reduced at the nuclear periphery. Moreover, RAG2 was distributed differently from RNA polymerase II and histone H3K4 trimethylation. Our data suggest that the nuclear periphery suppresses V(D)J recombination, at least in part, by segregating Tcrb alleles from RAG proteins.

Interdental osteotomies induce regional acceleratory phenomenon and accelerate orthodontic tooth movement.

PURPOSE: Although it has been revealed clinically that double-jaw orthognathic surgery induces a systemic increase in the baseline bone turnover and subsequently accelerates postoperative orthodontic tooth alignment, it is not clear whether less extensive osteotomy, such as interdental osteotomy, would be intensive enough to accelerate postoperative orthodontic tooth alignment. MATERIALS AND METHODS: Twelve adult male beagle dogs were randomly assigned to 2 groups. The sham control group (n = 6) received orthodontic tooth alignment of the maxillary incisors, and the experimental group (n = 6) received orthodontic tooth alignment of the maxillary incisors and interdental osteotomies between the maxillary third incisor and canine on both sides concurrent with the beginning of orthodontic tooth alignment. The duration of orthodontic tooth alignment was 3 months in both groups. Cone-beam computed tomography scans of the maxilla, dental casts, blood samples, and gingival crevicular fluid (GCF) of the maxillary incisors were taken immediately before orthodontic tooth alignment (T0) and 1 week (T1) and 1 (T2), 2 (T3), and 3 (T4) months after beginning orthodontic tooth alignment. They were analyzed for changes in the interdental alveolar bone gray scale (osteoporosity) of the maxillary incisors, irregularity index (rate of orthodontic tooth alignment), and bone-specific alkaline phosphatase (bALP, a biomarker of osteoblastic activity) and C-terminal telopeptide of type I collagen (ICTP, a biomarker for osteoclastic activity) in the serum and GCF samples from T0 to T4. The data were analyzed statistically for inter- and intragroup differences. RESULTS: The rate of orthodontic tooth alignment for the experimental group was significantly greater and twofold that of the sham control group at T1, T2, T3, and T4. The serum-bALP, serum-ICTP, GCF-bALP, GCF-ICTP, and osteoporosity remained similar from T0 to T4 in the sham control group. In contrast, GCF-bALP increased two- to threefold from T1 to T4, and GCF-ICTP increased fivefold at T1 and then gradually decreased from T2 to T4. The interdental osteoporosity significantly increased from T1 to T4, but the serum-bALP and serum-ICTP levels remained similar, without significant changes in the experimental group. CONCLUSIONS: Interdental osteotomy induced a regional, but not a systemic, acceleratory phenomenon and was intensive enough to accelerate postoperative orthodontic tooth alignment twofold. The intensity of the increase in the bone turnover rate and osteoporosity might depend on the extent of the osteotomy.

Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain type 2 immunity.

Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are required for miRNA activity. We generate Ago1, Ago3, and Ago4-deficient mice (Ago134Δ) and find AGO1/3/4 to be redundant for miRNA biogenesis, homeostasis, or function, a role that is carried out by AGO2. Instead, AGO1/3/4 regulate the expansion of type 2 immunity via precursor mRNA splicing in CD4+ T helper (Th) lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing, and in particular the isoforms of the gene Nisch, resulting in a dysregulated Nisch isoform ratio. This work uncouples AGO1, AGO3, and AGO4 from miRNA-mediated RNA interference, identifies an AGO3:SF3B3 complex in the nucleus, and reveals a mechanism by which AGO proteins regulate inflammatory diseases.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population.

Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

Regulation and Evolution of the RAG Recombinase.

The modular, noncontiguous architecture of the antigen receptor genes necessitates their assembly through V(D)J recombination. This program of DNA breakage and rejoining occurs during early lymphocyte development, and depends on the RAG1 and RAG2 proteins, whose collaborative endonuclease activity targets specific DNA motifs enriched in the antigen receptor loci. This essential gene shuffling reaction requires lymphocytes to traverse several developmental stages wherein DNA breakage is tolerated, while minimizing the expense to overall genome integrity. Thus, RAG activity is subject to stringent temporal and spatial regulation. The RAG proteins themselves also contribute autoregulatory properties that coordinate their DNA cleavage activity with target chromatin structure, cell cycle status, and DNA repair pathways. Even so, lapses in regulatory restriction of RAG activity are apparent in the aberrant V(D)J recombination events that underlie many lymphomas. In this review, we discuss the current understanding of the RAG endonuclease, its widespread binding in the lymphocyte genome, its noncleavage activities that restrain its enzymatic potential, and the growing evidence of its evolution from an ancient transposase.

Shhh! Silencing by microRNA-155.

Small RNAs mediate a diverse pot-pourri of post-transcriptional silencing mechanisms, ranging from 'classical' RNA interference (RNAi), to gene repression by microRNAs (miRNAs), to maintenance of genomic stability by repeat-associated small RNAs. Here, we review recent findings on the function of miR-155, particularly its roles in mammalian innate and adaptive immunity, viral infection and oncogenesis.

Long noncoding RNAs: implications for antigen receptor diversification.

Noncoding RNAs (ncRNAs), both small and large, have recently risen to prominence as surprisingly versatile regulators of gene expression. In fact, eukaryotic transcriptomes are rife with RNAs that do not code for protein, though the majority of these species remains wholly uncharacterized. The functional diversity among the mere handful of validated ncRNAs hints at the vast regulatory potential of these silent biomolecules. Though the act of noncoding transcription and the resultant ncRNAs do not directly produce proteins, they represent powerful means of gene control. Here we survey the accumulating literature on the myriad functions of long ncRNAs and emphasize one curious case of noncoding transcription at antigen receptor loci in lymphocytes.

Immunoglobulin somatic hypermutation.

The immunoglobulin (Ig) repertoire achieves functional diversification through several somatic alterations of the Ig locus. One of these processes, somatic hypermutation (SHM), deposits point mutations into the variable region of the Ig gene to generate higher-affinity variants. Activation-induced cytidine deaminase (AID) converts cytidine to uridine to initiate the hypermutation process. Error-prone versions of DNA repair are believed to then process these lesions into a diverse spectrum of point mutations. We review the current understanding of the molecular mechanisms and regulation of SHM, and also discuss emerging ideas which merit further exploration.

A mammalian microRNA expression atlas based on small RNA library sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.

Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, we set out to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. Our results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay.