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:
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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.
B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of ...Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sμ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Objectives
Inhibitors of bromodomain and extra terminal domain (BET) proteins are a new and growing class of anti‐cancer drugs, which decrease oncogene expression by targeting superenhancers. ...Antibody production is another physiological process relying on superenhancers, and it remains to be clarified whether potential immunomodulatory properties of BET inhibitors might impact humoral immunity and allergy.
Methods
We thus evaluated humoral immune responses and their Th2 context in vitro and in vivo in mice following treatment with the classical BET‐inhibitor JQ1. We quantified immunoglobulin (Ig) and antibody production by B cells either stimulated in vitro or obtained from immunised mice. JQ1 effects on class switching and activation‐induced deaminase loading were determined, together with modifications of B, T follicular helper (Tfh) and T helper 2 (Th2) populations. JQ1 was finally tested in B‐cell‐dependent models of immune disorders.
Results
Bromodomain and extra terminal domain inhibition reduced class switching, Ig expression on B cells and antibody secretion and was correlated with decreased numbers of Tfh cells. However, JQ1 strongly increased the proportion of GATA3+ Th2 cells and the secretion of corresponding cytokines. In a mouse allergic model of lung inflammation, JQ1 did not affect eosinophil infiltration or mucus production but enhanced Th2 cytokine production and aggravated clinical manifestations.
Conclusion
Altogether, BET inhibition thus interweaves intrinsic negative effects on B cells with a parallel complex reshaping of T‐cell polarisation which can increase type 2 cytokines and eventually promote B‐cell‐dependent immunopathology. These opposite and potentially hazardous immunomodulatory effects raise concerns for clinical use of BET inhibitors in patients with immune disorders.
While bromodomain and extra terminal domain inhibition reduces class switching, antibody secretion and T follicular helper cell number, this treatment increases the proportion of GATA3+ T helper 2 cells and the secretion of corresponding cytokines. In a mouse model of lung inflammation, JQ1 thus aggravates rather than improves clinical manifestations.
Many somatic genetic abnormalities have been identified in T-cell acute lymphoblastic leukemia (T-ALL) but each individual abnormality accounts for a small proportion of cases; therapeutic ...stratification consequently still relies on classical clinical markers. NOTCH1 and/or FBXW7 mutations both lead to activation of the NOTCH1 pathway and are among the most frequent mutations in T-ALL. We screened 141 adult diagnostic T-ALL samples from patients treated on either the Lymphoblastic Acute Leukemia in Adults (LALA)-94 (n = 87) or the GRAALL-2003 (n = 54) trials. In 88 cases (62%) there were demonstrated NOTCH1 mutations (42% heterodimerization HD, 10% HD+proline glutamate serine threonine PEST, 6% PEST, 2% juxtamembrane mutations, 2% transactivation domain TAD) and 34 cases (24%) had FBXW7 mutations (21 cases had both NOTCH1 and FBXW7 mutations); 40 cases (28%) were wild type for both. There was no significant correlation between NOTCH1 and/or FBXW7 mutations and clinico-biologic features. Median event-free survival (EFS) and overall survival (OS) were 36 versus 17 months (P = .01) and not reached versus 32 months (P = .004) in patients with NOTCH1 and/or FBXW7 mutations versus other patients, respectively. Multivariate analysis showed that the presence of NOTCH1/FBXW7 mutations was an independent good prognostic factor for EFS and OS (P = .02 and P = .01, respectively). These data demonstrate that NOTCH1 pathway activation by either NOTCH1 or FBXW7 mutation identifies a large group of patients with a favorable outcome that could justify individual therapeutic stratification for T-ALL.
T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently ...deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.
Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from ...illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRβ and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRβ (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) translocations. Our data also imply that oncogene activation takes place at a very immature stage of thymic development, when Dδ2-Dδ3/Dδ3-Jδ1 and Dβ-Jβ rearrangements occur, whereas the bulk leukemic maturation arrest occurs at a much later (cortical) stage. These observations have implications for T-ALL therapy, because the preleukemic early thymic clonogenic population needs to be eradicated and its disappearance monitored.
To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc(-/-) mice grafted with human umbilical cord blood CD34(+) hematopoietic progenitor cells (HPCs). Human ...prothymocytes developed in the murine bone marrow (BM) from multipotent CD34(++)CD38(lo)lineage(-) HPCs to CD34(++)CD7(+)CD2(-) pro-T1 cells that progressed in a Notch-dependent manner to CD34(+)CD7(++)CD2(+) pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34(+)CD7(lo) prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4-6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an "older" BM, prothymocyte differentiation was perturbed and resulted in CD34(+)CD7(lo) prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34(++)CD38(lo)lineage(-) HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34(+) HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc(-/-) mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.
The Common Variability Language (CVL) provides a well-structured mechanism to express variability and to relate this variability to any MOF-compliant model. This characteristic allows users to define ...the materialization of a given CVL resolution/configuration. Using variation points, it is possible to express and manipulate the links between the variability abstraction model and the base model. However, the meaning of a given variation point can vary according to the semantics of each domain. For example, a variation point that excludes an element in the base model can lead to further operations, like excluding other elements which were associated to the deleted element, or even to reassign references to another model element. Therefore, it is necessary to address this semantic variability in order to align the materialization semantics to the base model semantics. In this paper, we show how Kermeta can be used to easily implement and customize the semantics of the CVL's variation points, according to the semantics of the base model domain.
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