Somatic hypermutation mechanisms during lymphomagenesis and transformation.

B cells undergoing physiologically programmed or aberrant genomic alterations provide an opportune system to study the causes and consequences of genome mutagenesis. Activated B cells in germinal centers express activation-induced cytidine deaminase (AID) to accomplish physiological somatic hypermutation (SHM) of their antibody-encoding genes. In attempting to diversify their immunoglobulin (Ig) heavy- and light-chain genes, several B-cell clones successfully optimize their antigen-binding affinities. However, SHM can sometimes occur at non-Ig loci, causing genetic alternations that lay the foundation for lymphomagenesis, particularly diffuse large B-cell lymphoma. Thus, SHM acts as a double-edged sword, bestowing superb humoral immunity at the potential risk of initiating disease. We refer to off-target, non-Ig AID mutations - that are often but not always associated with disease - as aberrant SHM (aSHM). A key challenge in understanding SHM and aSHM is determining how AID targets and mutates specific DNA sequences in the Ig loci to generate antibody diversity and non-Ig genes to initiate lymphomagenesis. Herein, we discuss some current advances regarding the regulation of AID's DNA mutagenesis activity in B cells.

Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression.

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner.

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N6-methyladenosine (m6A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m6A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m6A methylated in ILC2s. Targeted m6A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m6A for ILC2 responses.

RNA-regulatory exosome complex suppresses an apoptotic program to confer erythroid progenitor cell survival in vivo.

The RNA-regulatory exosome complex (EC) posttranscriptionally and cotranscriptionally processes and degrades RNAs in a context-dependent manner. Although the EC functions in diverse cell types, its contributions to stem and progenitor cell development are not well understood. Previously, we demonstrated that the transcriptional regulator of erythrocyte development, GATA1, represses EC subunit genes, and the EC maintains erythroid progenitors in vitro. To determine if this mechanism operates in vivo, we used the hematopoietic-specific Vav1-Cre and "conditional by inversion" mouse system to ablate Exosc3, encoding an EC structural subunit. Although Exosc3C/C Cre+ embryos developed normally until embryonic day 14.5, Exosc3 ablation was embryonic lethal and severely reduced erythromyeloid progenitor activity. RNA sequencing analysis of Exosc3-ablated burst-forming unit-erythroid revealed elevated transcripts encoding multiple proapoptotic factors, and the mutant erythroid progenitors exhibited increased apoptosis. We propose that the EC controls an ensemble of apoptosis-regulatory RNAs, thereby promoting erythroid progenitor survival and developmental erythropoiesis in vivo.

Identification of RNA-DNA Hybrids Associated with R-Loops at the IgH Switch Sequence in Activated B Cells.

During transcription and replication, R-loops that contain RNA-DNA hybrids are generated across numerous genomic loci and contribute to many biological events. Using S9.6, a monoclonal antibody against RNA-DNA hybrids, accelerated the study of R-loop biology. An outpouring of recent studies has implicated various contributions of R-loop in physiological cellular functions. Earlier studies using nondenaturing sodium bisulfite probing also supported existence of DNA-RNA hybrids formation in mammalian cells. In activated B cells, RNA-DNA hybrids formation at IgH gene locus of B cells is crucial for class switch recombination that ensure the proper effector function of the antibody. Here, we describe the identification of R-loops associated with the IgH locus using RNA-DNA hybrid immunoprecipitation sequencing and nondenaturing sodium bisulfite probing. This will be helpful for future studies of R-loops status on whole genome as well as on IgH locus in B cells.

RNA exosome drives early B cell development via noncoding RNA processing mechanisms.

B cell development is linked to successful V(D)J recombination, allowing B cell receptor expression and ultimately antibody secretion for adaptive immunity. Germline noncoding RNAs (ncRNAs) are produced at immunoglobulin (Ig) loci during V(D)J recombination, but their function and posttranscriptional regulation are incompletely understood. Patients with trichohepatoenteric syndrome, characterized by RNA exosome pathway component mutations, exhibit lymphopenia, thus demonstrating the importance of ncRNA surveillance in B cell development in humans. To understand the role of RNA exosome in early B cell development in greater detail, we generated mouse models harboring a B cell-specific cre allele (Mb1cre), coupled to conditional inversion-deletion alleles of one RNA exosome core component (Exosc3) or RNase catalytic subunits (Exosc10 or Dis3). We noticed increased expression of RNA exosome subunits during V(D)J recombination, whereas a B cell developmental blockade at the pro-B cell stage was observed in the different knockout mice, overlapping with a lack of productive rearrangements of VDJ genes at the Ig heavy chain (Igh). This unsuccessful recombination prevented differentiation into pre-B cells, with accumulation of ncRNAs and up-regulation of the p53 pathway. Introduction of a prearranged Igh VDJ allele partly rescued the pre-B cell population in Dis3-deficient cells, although V-J recombination defects were observed at Ig light chain kappa (Igκ), preventing subsequent B cell development. These observations demonstrated that the RNA exosome complex is important for Igh and Igκ recombination and establish the relevance of RNA processing for optimal diversification at these loci during B cell development.

Mechanism of noncoding RNA-associated N6-methyladenosine recognition by an RNA processing complex during IgH DNA recombination.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SµGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3' end processing of SµGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SµGLT. Direct suppression of m6A modification of SµGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.

A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

Proteasomal Regulation of Mammalian SPT16 in Controlling Transcription.

FACT (facilitates chromatin transcription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer.

Noncoding RNA processing by DIS3 regulates chromosomal architecture and somatic hypermutation in B cells.

Noncoding RNAs are exquisitely titrated by the cellular RNA surveillance machinery for regulating diverse biological processes. The RNA exosome, the predominant 3' RNA exoribonuclease in mammalian cells, is composed of nine core and two catalytic subunits. Here, we developed a mouse model with a conditional allele to study the RNA exosome catalytic subunit DIS3. In DIS3-deficient B cells, integrity of the immunoglobulin heavy chain (Igh) locus in its topologically associating domain is affected, with accumulation of DNA-associated RNAs flanking CTCF-binding elements, decreased CTCF binding to CTCF-binding elements and disorganized cohesin localization. DIS3-deficient B cells also accumulate activation-induced cytidine deaminase-mediated asymmetric nicks, altering somatic hypermutation patterns and increasing microhomology-mediated end-joining DNA repair. Altered mutation patterns and Igh architectural defects in DIS3-deficient B cells lead to decreased class-switch recombination but increased chromosomal translocations. Our observations of DIS3-mediated architectural regulation at the Igh locus are reflected genome wide, thus providing evidence that noncoding RNA processing is an important mechanism for controlling genome organization.

Purification of Murine IL-10 + B Cells for Analyses of Biological Functions and Transcriptomics.

B10 cells are the most frequently investigated subset of Breg cells, capable of suppressing immunity through the expression of the immunosuppressive cytokine IL-10. B10 cells are enriched in phenotypically diverse B-cell subsets. Recently, CD9 was identified as a marker of B10 cells in mice (human B10 cells have a separate set of markers that do not overlap with murine B10 cells). Together with a combination of other B10 markers, CD9 can be used to distinguish both mature and immature B10 cells from nonregulatory B cells and support selective purification of B10 cells. Here we provide five methods for the characterization and activity evaluation of CD9+ B cells. The first method is used for the preparation of leukocytes, the second and third are used for the characterization of CD9+ B cells, while the last two methods serve to evaluate CD9+ B-cell activities. Finally, we detail the purification of RNA from B10 cells and the performance of transcriptomic assays.

Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling.

Fine-tuned gene expression is crucial for neurodevelopment. The gene expression program is tightly controlled at different levels, including RNA decay. N6-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for brain development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay contributes to brain development is largely unknown. Here, we show that Exosc10, a RNA exonuclease subunit of the RNA exosome complex, is indispensable for forebrain development. We report that cortical cells undergo overt apoptosis, culminating in cortical agenesis upon conditional deletion of Exosc10 in mouse cortex. Mechanistically, Exosc10 directly binds and degrades transcripts of the P53 signaling-related genes, such as Aen and Bbc3. Overall, our findings suggest a crucial role for Exosc10 in suppressing the P53 pathway, in which the rapid turnover of the apoptosis effectors Aen and Bbc3 mRNAs is essential for cell survival and normal cortical histogenesis.

ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma.

Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.

CD8+ T-cell-Mediated Immunoediting Influences Genomic Evolution and Immune Evasion in Murine Gliomas.

PURPOSE: Cancer immunoediting shapes tumor progression by the selection of tumor cell variants that can evade immune recognition. Given the immune evasion and intratumor heterogeneity characteristic of gliomas, we hypothesized that CD8+ T cells mediate immunoediting in these tumors. EXPERIMENTAL DESIGN: We developed retrovirus-induced PDGF+ Pten -/- murine gliomas and evaluated glioma progression and tumor immunogenicity in the absence of CD8+ T cells by depleting this immune cell population. Furthermore, we characterized the genomic alterations present in gliomas that developed in the presence and absence of CD8+ T cells. RESULTS: Upon transplantation, gliomas that developed in the absence of CD8+ T cells engrafted poorly in recipients with intact immunity but engrafted well in those with CD8+ T-cell depletion. In contrast, gliomas that developed under pressure from CD8+ T cells were able to fully engraft in both CD8+ T-cell-depleted mice and immunocompetent mice. Remarkably, gliomas developed in the absence of CD8+ T cells exhibited increased aneuploidy, MAPK pathway signaling, gene fusions, and macrophage/microglial infiltration, and showed a proinflammatory phenotype. MAPK activation correlated with macrophage/microglia recruitment in this model and in the human disease. CONCLUSIONS: Our studies indicate that, in these tumor models, CD8+ T cells influence glioma oncogenic pathways, tumor genotype, and immunogenicity. This suggests immunoediting of immunogenic tumor clones through their negative selection by CD8+ T cells during glioma formation.

Effects of senataxin and RNA exosome on B-cell chromosomal integrity.

Loss of function of senataxin (SETX), a bona-fide RNA/DNA helicase, is associated with neuronal degeneration leading to Ataxia and Ocular Apraxia (AOA) in human patients. SETX is proposed to promote transcription termination, DNA replication, DNA repair, and to unwind deleterious RNA:DNA hybrids in the genome. In all the above-mentioned mechanisms, SETX unwinds transcription complex-associated nascent RNA which is then degraded by the RNA exosome complex. Here we have used B cells isolated from a SETX mutant mouse model and compared genomic instability and immunoglobulin heavy chain locus (IgH) class switch recombination (CSR) to evaluate aberrant and programmed genomic rearrangements, respectively. Similar to RNA exosome mutant primary B cells, SETX mutant primary B cells display genomic instability but a modest decrease in efficiency of CSR. Furthermore, knockdown of Setx mRNAs from CH12-F3 B-cell lines leads to a defect in IgA CSR and accumulation of aberrant patterns of mutations in IgH switch sequences. Given that SETX mutant mice do not recapitulate the AOA neurodegenerative phenotype, it is possible that some aspects of SETX biology are rescued by redundant helicases in mice. Overall, our study provides new insights into the role of the SETX/RNA exosome axis in suppressing genomic instability so that programmed DNA breaks are properly orchestrated.

Noncoding RNA transcription alters chromosomal topology to promote isotype-specific class switch recombination.

B cells undergo two types of genomic alterations to increase antibody diversity: introduction of point mutations into immunoglobulin heavy- and light-chain (IgH and IgL) variable regions by somatic hypermutation (SHM) and alteration of antibody effector functions by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). SHM and CSR require the B cell-specific activation-induced cytidine deaminase (AID) protein, the transcription of germline noncoding RNAs, and the activity of the 3' regulatory region (3'RR) super-enhancer. Although many transcription regulatory elements (e.g., promoters and enhancers) reside inside the IgH and IgL sequences, the question remains whether clusters of regulatory elements outside IgH control CSR. Using RNA exosome-deficient mouse B cells where long noncoding RNAs (lncRNAs) are easily detected, we identified a cluster of three RNA-expressing elements that includes lncCSRIgA (that expresses lncRNA-CSRIgA). B cells isolated from a mouse model lacking lncRNA-CSRIgA transcription fail to undergo normal levels of CSR to IgA both in B cells of the Peyer's patches and grown in ex vivo culture conditions. lncRNA-CSRIgA is expressed from an enhancer site (lncCSRIgA ) to facilitate the recruitment of regulatory proteins to a nearby CTCF site (CTCFlncCSR) that alters the chromosomal interactions inside the TADlncCSRIgA and long-range interactions with the 3'RR super-enhancer. Humans with IgA deficiency show polymorphisms in the lncCSRIgA locus compared with the normal population. Thus, we provide evidence for an evolutionarily conserved topologically associated domain (TADlncCSRIgA) that coordinates IgA CSR in Peyer's patch B cells through an lncRNA (lncRNA-CSRIgA) transcription-dependent mechanism.

Regulation of long non-coding RNAs and genome dynamics by the RNA surveillance machinery.

Much of the mammalian genome is transcribed, generating long non-coding RNAs (lncRNAs) that can undergo post-transcriptional surveillance whereby only a subset of the non-coding transcripts is allowed to attain sufficient stability to persist in the cellular milieu and control various cellular functions. Paralleling protein turnover by the proteasome complex, lncRNAs are also likely to exist in a dynamic equilibrium that is maintained through constant monitoring by the RNA surveillance machinery. In this Review, we describe the RNA surveillance factors and discuss the vital role of lncRNA surveillance in orchestrating various biological processes, including the protection of genome integrity, maintenance of pluripotency of embryonic stem cells, antibody-gene diversification, coordination of immune cell activation and regulation of heterochromatin formation. We also discuss examples of human diseases and developmental defects associated with the failure of RNA surveillance mechanisms, further highlighting the importance of lncRNA surveillance in maintaining cell and organism functions and health.

Biology of RNA Surveillance in Development and Disease.

The 'RNA world', in which RNA molecules stored information and acquired enzymatic properties, has been proposed to have preceded organism life. RNA is now recognized for its central role in biology, with accumulating evidence implicating coding and noncoding (nc)RNAs in myriad mechanisms regulating cellular physiology and disequilibrium in transcriptomes resulting in pathological conditions. Nascently synthesized RNAs are subjected to stringent regulation by sophisticated RNA surveillance pathways. In this review, we integrate these pathways from a developmental viewpoint, proposing RNA surveillance as the convergence of mechanisms that ensure the exact titration of RNA molecules in a spatiotemporally controlled manner, leading to development without the onset of pathological conditions, including cancer.