Nuclear Proximity of Mtr4 to RNA Exosome Restricts DNA Mutational Asymmetry.

The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.

Fam72a enforces error-prone DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

Detecting Rare AID-Induced Mutations in B-Lineage Oncogenes from High-Throughput Sequencing Data Using the Detection of Minor Variants by Error Correction Method.

In B-lineage cells, the cytidine deaminase AID not only generates somatic mutations to variable regions of Ig genes but also inflicts, at a lower frequency, mutations to several non-Ig genes named AID off-targets, which include proto-oncogenes. High-throughput sequencing should be in principle the method of choice to detect and document these rare nucleotide substitutions. So far, high-throughput sequencing-based methods are impaired by a global sequencing error rate that usually covers the real mutation rate of AID off-target genes in activated B cells. We demonstrate the validity of a per-base background subtraction method called detection of minor variants by error correction (DeMinEr), which uses deep sequencing data from mutated and nonmutated samples to correct the substitution frequency at each nucleotide position along the sequenced region. Our DeMinEr method identifies somatic mutations at a frequency down to 0.02% at any nucleotide position within two off-target genes: Cd83 and Bcl6 Biological models and control conditions such as AID- and UNG-deficient mice validate the specificity and the sensitivity of our method. The high resolution and robustness of DeMinEr enable us to document fine effects such as age-dependent accumulation of mutations in these oncogenes in the mouse.

Sequential activation and distinct functions for distal and proximal modules within the IgH 3' regulatory region.

As a master regulator of functional Ig heavy chain (IgH) expression, the IgH 3' regulatory region (3'RR) controls multiple transcription events at various stages of B-cell ontogeny, from newly formed B cells until the ultimate plasma cell stage. The IgH 3'RR plays a pivotal role in early B-cell receptor expression, germ-line transcription preceding class switch recombination, interactions between targeted switch (S) regions, variable region transcription before somatic hypermutation, and antibody heavy chain production, but the functional ranking of its different elements is still inaccurate, especially that of its evolutionarily conserved quasi-palindromic structure. By comparing relevant previous knockout (KO) mouse models (3'RR KO and hs3b-4 KO) to a novel mutant devoid of the 3'RR quasi-palindromic region (3'PAL KO), we pinpointed common features and differences that specify two distinct regulatory entities acting sequentially during B-cell ontogeny. Independently of exogenous antigens, the 3'RR distal part, including hs4, fine-tuned B-cell receptor expression in newly formed and naïve B-cell subsets. At mature stages, the 3'RR portion including the quasi-palindrome dictated antigen-dependent locus remodeling (global somatic hypermutation and class switch recombination to major isotypes) in activated B cells and antibody production in plasma cells.

The Eµ enhancer region influences H chain expression and B cell fate without impacting IgVH repertoire and immune response in vivo.

The IgH intronic enhancer region Eµ is a combination of both a 220-bp core enhancer element and two 310-350-bp flanking scaffold/matrix attachment regions named MARsEµ. In the mouse, deletion of the core-enhancer Eµ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eµ region (core plus MARsEµ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro-B cell stage, Eµ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre-B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igµ H chain expression was decreased in Eµ-deficient pre-B cells, we propose that modification of B cell homeostasis in deficient animals was caused by "weak" pre-B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eµ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eµ region does not influence VH segment usage but ensures efficient Igµ-chain expression required for strong signaling through pre-B cells and newly formed BCRs and thus participates in B cell inflow and fate.

Long-range oncogenic activation of Igh-c-myc translocations by the Igh 3' regulatory region.

B-cell malignancies, such as human Burkitt's lymphoma, often contain translocations that link c-myc or other proto-oncogenes to the immunoglobulin heavy chain locus (IgH, encoded by Igh). The nature of elements that activate oncogenes within such translocations has been a long-standing question. Translocations within Igh involve DNA double-strand breaks initiated either by the RAG1/2 endonuclease during variable, diversity and joining gene segment (V(D)J) recombination, or by activation-induced cytidine deaminase (AID, also known as AICDA) during class switch recombination (CSR). V(D)J recombination in progenitor B (pro-B) cells assembles Igh variable region exons upstream of mu constant region (Cmu) exons, which are the first of several sets of C(H) exons ('C(H) genes') within a C(H) locus that span several hundred kilobases (kb). In mature B cells, CSR deletes Cmu and replaces it with a downstream C(H) gene. An intronic enhancer (iEmu) between the variable region exons and Cmu promotes V(D)J recombination in developing B cells. Furthermore, the Igh 3' regulatory region (Igh3'RR) lies downstream of the C(H) locus and modulates CSR by long-range transcriptional enhancement of C(H) genes. Transgenic mice bearing iEmu or Igh3'RR sequences fused to c-myc are predisposed to B lymphomas, demonstrating that such elements can confer oncogenic c-myc expression. However, in many B-cell lymphomas, Igh-c-myc translocations delete iEmu and place c-myc up to 200 kb upstream of the Igh3'RR. Here we address the oncogenic role of the Igh3'RR by inactivating it in two distinct mouse models for B-cell lymphoma with Igh-c-myc translocations. We show that the Igh3'RR is dispensable for pro-B-cell lymphomas with V(D)J recombination-initiated translocations, but is required for peripheral B-cell lymphomas with CSR-associated translocations. As the Igh3'RR is not required for CSR-associated Igh breaks or Igh-c-myc translocations in peripheral B-cell lymphoma progenitors, we conclude that this regulatory region confers oncogenic activity by long-range and developmental stage-specific activation of translocated c-myc genes.

Enhancers located in heavy chain regulatory region (hs3a, hs1,2, hs3b, and hs4) are dispensable for diversity of VDJ recombination.

V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple IgH cis-regulatory elements control B-cell ontogeny. IGCR1 (intergenic control region 1), the DQ52 promoter/enhancer, and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination, whereas there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here, we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B-cell differentiation. In contrast, the earliest antigen-independent steps of B-cell ontogeny would be under the control of only the upstream Cmu elements of the locus.

A dual function for the chromatin organizer Special A-T rich Binding Protein 1 in B-lineage cells.

SATB1 (Special A-T rich Binding protein 1) is a cell type-specific factor that regulates the genetic network in developing T cells and neurons. In T cells, SATB1 is required for lineage commitment, VDJ recombination, development and maturation. Considering that its expression varies during B-cell differentiation, the involvement of SATB1 needs to be clarified in this lineage. Using a KO mouse model in which SATB1 was deleted from the pro-B-cell stage, we examined the consequences of SATB1 deletion in naive and activated B-cell subsets. Our model indicates first, unlike its essential function in T cells, that SATB1 is dispensable for B-cell development and the establishment of a broad IgH repertoire. Second, we show that SATB1 exhibits an ambivalent function in mature B cells, acting sequentially as a positive and negative regulator of Ig gene transcription in naive and activated cells, respectively. Third, our study indicates that the negative regulatory function of SATB1 in B cells extends to the germinal center response, in which this factor limits somatic hypermutation of Ig genes.

The IgH Eµ-MAR regions promote UNG-dependent error-prone repair to optimize somatic hypermutation.

Intoduction: Two scaffold/matrix attachment regions (5'- and 3'-MARsEµ ) flank the intronic core enhancer (cEµ) within the immunoglobulin heavy chain locus (IgH). Besides their conservation in mice and humans, the physiological role of MARsEµ is still unclear and their involvement in somatic hypermutation (SHM) has never been deeply evaluated. Methods: Our study analyzed SHM and its transcriptional control in a mouse model devoid of MARsEµ , further combined to relevant models deficient for base excision repair and mismatch repair. Results: We observed an inverted substitution pattern in of MARsEµ -deficient animals: SHM being decreased upstream from cEµ and increased downstream of it. Strikingly, the SHM defect induced by MARsEµ -deletion was accompanied by an increase of sense transcription of the IgH V region, excluding a direct transcription-coupled effect. Interestingly, by breeding to DNA repair-deficient backgrounds, we showed that the SHM defect, observed upstream from cEµ in this model, was not due to a decrease in AID deamination but rather the consequence of a defect in base excision repair-associated unfaithful repair process. Discussion: Our study pointed out an unexpected "fence" function of MARsEµ regions in limiting the error-prone repair machinery to the variable region of Ig gene loci.

Deletion of the α immunoglobulin chain membrane-anchoring region reduces but does not abolish IgA secretion.

Class switching and plasma cell differentiation occur at a high level within all mucosa-associated lymphoid tissues. The different classes of membrane immunoglobulin heavy chains are associated with the Igα/Igß heterodimer within the B-cell receptor (BCR). Whether BCR isotypes convey specific signals adapted to the corresponding differentiation stages remains debated but IgG and IgA membranes have been suggested to promote plasma cell differentiation. We investigated the impact of blocking expression of the IgA-class BCR through a 'αΔtail' targeted mutation, deleting the Cα immunoglobulin gene membrane exon. This allowed us to evaluate to what extent class switching and plasma cell differentiation can be concurrent processes, allowing some αΔtail(+/+) B cells with an IgM BCR to directly differentiate into IgA plasma cells and yield serum secreted IgA in spite of the absence of membrane IgA(+) B lymphocytes. By contrast, in secretions the secretory IgA was very low, indicating that J-chain-positive plasma cells producing secretory IgA overwhelmingly differentiate from previously class-switched membrane IgA(+) memory B cells. In addition, although mucosa-associated lymphoid tissues are a major site for plasma cell accumulation, αΔtail(+/+) mice showed that the gut B-cell lineage homeostasis is not polarized toward plasma cell differentiation through a specific influence of the membrane IgA BCR.

Genomic deletion of the whole IgH 3' regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes.

The immunoglobulin heavy chain locus (IgH) undergoes multiple changes along B-cell differentiation. In progenitor B cells, V(D)J assembly allows expression of µ heavy chains. In mature B cells, class switch recombination may replace the expressed constant (C)µ gene with a downstream C(H) gene. Finally, plasma cell differentiation strongly boosts IgH transcription. How the multiple IgH transcriptional enhancers tune these changes is unclear. Here we demonstrate that deletion of the whole IgH 3' regulatory region (3'RR) allows normal maturation until the stage of IgM/IgD expressing lymphocytes, but nearly abrogates class switch recombination to all C(H) genes. Although plasma cell numbers are unaffected, we reveal the role of the 3'RR into the transcriptional burst normally associated with plasma cell differentiation. Our study shows that transcriptional changes and recombinations occurring after antigen-encounter appear mainly controlled by the 3'RR working as a single functional unit.

In vivo redundant function of the 3' IgH regulatory element HS3b in the mouse.

In the mouse, the regulatory region located at the 3' end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igmicro-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

Increased B cell proliferation and reduced Ig production in DREAM transgenic mice.

DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum Ig levels. In vitro assays show that reduced Ig secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation, class switch recombination, or Ig transcription. Surprisingly, transgenic B cells show an accelerated entry in cell division. Transcriptomic analysis of transgenic B cells revealed that hyperproliferative B cell response could be correlated with a reduced expression of Klf9, a cell-cycle regulator. Pulse-chase experiments demonstrated that the defect in Ig production is associated with reduced translation rather than with increased protein degradation. Importantly, transgenic B cells showed reduced expression of the Eif4g3 gene, which encodes a protein related to protein translation. Our results disclose, to our knowledge, a novel function of DREAM in proliferation and Ig synthesis in B lymphocytes.

Contribution of Immunoglobulin Enhancers to B Cell Nuclear Organization.

B cells undergo genetic rearrangements at immunoglobulin gene (Ig) loci during B cell maturation. First V(D)J recombination occurs during early B cell stages followed by class switch recombination (CSR) and somatic hypermutation (SHM) which occur during mature B cell stages. Given that RAG1/2 induces DNA double strand breaks (DSBs) during V(D)J recombination and AID (Activation-Induced Deaminase) leads to DNA modifications (mutations during SHM or DNA DSBs during CSR), it is mandatory that IgH rearrangements be tightly regulated to avoid any mutations or translocations within oncogenes. Ig loci contain various cis-regulatory elements that are involved in germline transcription, chromatin modifications or RAG/AID recruitment. Ig cis-regulatory elements are increasingly recognized as being involved in nuclear positioning, heterochromatin addressing and chromosome loop regulation. In this review, we examined multiple data showing the critical interest of studying Ig gene regulation at the whole nucleus scale. In this context, we highlighted the essential function of Ig gene regulatory elements that now have to be considered as nuclear organizers in B lymphocytes.

The IgH 3' regulatory region and its implication in lymphomagenesis.

The 3' regulatory region (3'RR) located downstream of the IgH gene is the master element that controls class switch recombination and sustains high-level transcription at the plasma-cell stage. This latter role suggests that the 3'RR may be involved in oncogene deregulation during the frequent IgH translocation events associated with B-cell malignancies. A convincing demonstration of the essential contribution of 3'RR in lymphomagenesis has been provided by transgenic animal models. The mouse 3'RR shares a strong structural homology with the regulatory regions located downstream of each human Cα gene. Mouse models exploring the role of the 3'RR in B-cell physiology and in malignancies should provide useful indications about the pathophysiology of human cell lymphocyte proliferation.

Panorama of stepwise involvement of the IgH 3' regulatory region in murine B cells.

Among the multiple events leading to immunoglobulin (Ig) expression in B cells, stepwise activation of the Ig heavy chain locus (IgH) is of critical importance. Transcription regulation of the complex IgH locus has always been an interesting viewpoint to unravel the multiple and complex events required for IgH expression. First, regulatory germline transcripts (GLT) assist DNA remodeling events such as VDJ recombination, class switch recombination (CSR) and somatic hypermutation (SHM). Second, productive spliced transcripts restrict heavy chain protein expression associated either with the surface receptor of developing B cells or secreted in large amounts in plasma cells. One main transcriptional regulator for IgH lies at its 3' extremity and includes both a set of enhancers grouped in a large 3' regulatory region (3'RR) and a cluster of 3'CTCF-binding elements (3'CBEs). In this focused review, we will preferentially refer to evidence reported for the murine endogenous IgH locus, whether it is wt or carries deletions or insertions within the IgH 3' boundary and associated regulatory region.

Physiological and druggable skipping of immunoglobulin variable exons in plasma cells.

The error-prone V(D)J recombination process generates considerable amounts of nonproductive immunoglobulin (Ig) pre-mRNAs. We recently demonstrated that aberrant Ig chains lacking variable (V) domains can be produced after nonsense-associated altered splicing (NAS) events. Remarkably, the expression of these truncated Ig polypeptides heightens endoplasmic reticulum stress and shortens plasma cell (PC) lifespan. Many questions remain regarding the molecular mechanisms underlying this new truncated Ig exclusion (TIE-) checkpoint and its restriction to the ultimate stage of B-cell differentiation. To address these issues, we evaluated the extent of NAS of Ig pre-mRNAs using an Ig heavy chain (IgH) knock-in model that allows for uncoupling of V exon skipping from TIE-induced apoptosis. We found high levels of V exon skipping in PCs compared with B cells, and this skipping was correlated with a biallelic boost in IgH transcription during PC differentiation. Chromatin analysis further revealed that the skipped V exon turned into a pseudo-intron. Finally, we showed that hypertranscription of Ig genes facilitated V exon skipping upon passive administration of splice-switching antisense oligonucleotides (ASOs). Thus, V exon skipping is coupled to transcription and increases as PC differentiation proceeds, likely explaining the late occurrence of the TIE-checkpoint and opening new avenues for ASO-mediated strategies in PC disorders.

The IgH locus 3' cis-regulatory super-enhancer co-opts AID for allelic transvection.

Immunoglobulin heavy chain (IgH) alleles have ambivalent relationships: they feature both allelic exclusion, ensuring monoallelic expression of a single immunoglobulin (Ig) allele, and frequent inter-allelic class-switch recombination (CSR) reassembling genes from both alleles. The IgH locus 3' regulatory region (3'RR) includes several transcriptional cis-enhancers promoting activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) and CSR, and altogether behaves as a strong super-enhancer. It can also promote deregulated expression of translocated oncogenes during lymphomagenesis. Besides these rare, illegitimate and pathogenic interactions, we now show that under physiological conditions, the 3'RR super-enhancer supports not only legitimate cis- , but also trans-recruitment of AID, contributing to IgH inter-allelic proximity and enabling the super-enhancer on one allele to stimulate biallelic SHM and CSR. Such inter-allelic activating interactions define transvection, a phenomenon well-known in drosophila but rarely observed in mammalian cells, now appearing as a unique feature of the IgH 3'RR super-enhancer.

The IgH 3' regulatory region governs µ chain transcription in mature B lymphocytes and the B cell fate.

We report that the IgH 3' regulatory region (3'RR) has no role on µ chain transcription and pre-BCR expression in B cell progenitors. In contrast, analysis of heterozygous IgH aΔ3'RR/bwt mice indicated that the 3'RR controls µ chain transcripts in mature splenocytes and impacts membrane IgM density without obvious effect on BCR signals (colocalisation with lipid rafts and phosphorylation of Erk and Akt after BCR crosslinking). Deletion of the 3'RR modulates the B cell fate to less marginal zone B cells. In conclusion, the 3'RR is dispensable for pre-BCR expression and necessary for optimal commitments toward the marginal zone B cell fate. These results reinforce the concept of a dual regulation of the IgH locus transcription and accessibility by 5' elements at immature B cell stages, and by the 3'RR as early as the resting mature B cell stage and then along further activation and differentiation.

The IgH 3' regulatory region controls somatic hypermutation in germinal center B cells.

Interactions with cognate antigens recruit activated B cells into germinal centers where they undergo somatic hypermutation (SHM) in V(D)J exons for the generation of high-affinity antibodies. The contribution of IgH transcriptional enhancers in SHM is unclear. The Eµ enhancer upstream of Cµ has a marginal role, whereas the influence of the IgH 3' regulatory region (3'RR) enhancers (hs3a, hs1,2, hs3b, and hs4) is controversial. To clarify the latter issue, we analyzed mice lacking the whole 30-kb extent of the IgH 3'RR. We show that SHM in VH rearranged regions is almost totally abrogated in 3'RR-deficient mice, whereas the simultaneous Ig heavy chain transcription rate is only partially reduced. In contrast, SHM in κ light chain genes remains unaltered, acquitting for any global SHM defect in our model. Beyond class switch recombination, the IgH 3'RR is a central element that controls heavy chain accessibility to activation-induced deaminase modifications including SHM.