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Phosphoinositide-3 kinase (PI3K) signaling is important for the survival of numerous cell types and class IA of PI3K is specifically required for the development of B cells but not for T cell development. Here, we show that class IA PI3K-mediated signals induce the expression of the transcription factor Pax5, which plays a central role in B cell commitment and differentiation by activating the expression of central B cell-specific signaling proteins such as SLP-65 and CD19. Defective class IA PI3K function leads to reduction in Pax5 expression and prevents B cell development beyond the stage expressing the precursor B cell receptor (pre-BCR). Investigating the mechanism of PI3K-induced Pax5 expression revealed that it involves a network of transcription factors including FoxO1 and Irf4 that directly binds to the Pax5 gene. Together, our results suggest that PI3K signaling links survival and differentiation of developing B cells with B cell identity and that decreased PI3K activity in pre-B cells results in reduced Pax5 expression and lineage plasticity.

Accumulating evidence suggests that Pax5 plays essential roles in B cell lineage commitment. However, molecular mechanisms of B cell-specific expression of Pax5 are not fully understood. Here, we applied insertional chromatin immunoprecipitation (iChIP) combined with stable isotope labeling using amino acids in cell culture (SILAC) (iChIP-SILAC) to direct identification of proteins interacting with the promoter region of the endogenous single-copy chicken Pax5 gene. By comparing B cells with macrophage-like cells trans-differentiated by ectopic expression of C/EBPβ, iChIP-SILAC detected B cell-specific interaction of a nuclear protein, Thy28/Thyn1, with the Pax5 1A promoter. Trans-differentiation of B cells into macrophage-like cells caused down-regulation of Thy28 expression. Loss-of-function of Thy28 induced decrease in Pax5 expression and recruitment of myosin-9 (MYH9), one of Thy28-interacting proteins, to the Pax5 1A promoter. Loss-of-function of MYH9 also induced decrease in Pax5 expression. Thus, our analysis revealed that Thy28 is functionally required for B cell-specific expression of Pax5 via recruitment of MYH9 to the Pax5 locus in chicken B cells.

Background Histone Mark Modifications (HMs) are crucial actors in gene regulation, as they actively remodel chromatin to modulate transcriptional activity: aberrant combinatorial patterns of HMs have been connected with several diseases, including cancer. HMs are, however, reversible modifications: understanding their role in disease would allow the design of ‘epigenetic drugs’ for specific, non-invasive treatments. Standard statistical techniques were not entirely successful in extracting representative features from raw HM signals over gene locations. On the other hand, deep learning approaches allow for effective automatic feature extraction, but at the expense of model interpretation. Results Here, we propose ShallowChrome, a novel computational pipeline to model transcriptional regulation via HMs in both an accurate and interpretable way. We attain state-of-the-art results on the binary classification of gene transcriptional states over 56 cell-types from the REMC database, largely outperforming recent deep learning approaches. We interpret our models by extracting insightful gene-specific regulative patterns, and we analyse them for the specific case of the PAX5 gene over three differentiated blood cell lines. Finally, we compare the patterns we obtained with the characteristic emission patterns of ChromHMM, and show that ShallowChrome is able to coherently rank groups of chromatin states w.r.t. their transcriptional activity. Conclusions In this work we demonstrate that it is possible to model HM-modulated gene expression regulation in a highly accurate, yet interpretable way. Our feature extraction algorithm leverages on data downstream the identification of enriched regions to retrieve gene-wise, statistically significant and dynamically located features for each HM. These features are highly predictive of gene transcriptional state, and allow for accurate modeling by computationally efficient logistic regression models. These models allow a direct inspection and a rigorous interpretation, helping to formulate quantifiable hypotheses.

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair 1 . Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression 2 , a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells 3 . Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1 , MYC , RhoH / TTF ( ARHH ) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5′ untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks 4 , 5 , 6 . By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.

Epigenetic changes have major role in the normal development and programming of gene expression. Aberrant methylation results in carcinogenesis. The primary objective of our study is to determine whether primary tumor tissue and cultured tumor cells in 2D and 3D tissue culture systems have the same methylation signature for PAX5, TMPRSS2, and SBDS. These findings will play an important role in developing in vitro model system to understand the effect of methylation inhibitors on primary tumor tissue. In a previous study PAX5, TMPRSS2, and SBDS genes that we are investigating were reported to be methylated more than 60% in breast cancer and malignant melanoma cell lines. However, these genes have never been studied in primary tumor tissues. Thus, primary tumor tissues of breast cancer and malignant melanoma were first grown in 2D and 3D cultures. Then these two types of tumor tissues and their 2D and 3D cultures were investigated for changes considering methylation levels in PAX5, TMPRSS2, and SBDS genes using real-time polymerase chain reaction. No differences were observed in the primary tissues and culture systems for both PAX5 and TMPRSS2 in malignant melanoma tissues. We found that PAX5 gene was an efficient marker to measure the effects of methylation inhibitors for in vitro systems for malignant melanoma tissue.

Pax5 controls the identity and development of B cells by repressing lineage‐inappropriate genes and activating B‐cell‐specific genes. Here, we used genome‐wide approaches to identify Pax5 target genes in pro‐B and mature B cells. In these cell types, Pax5 bound to 40% of the cis ‐regulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro‐B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5‐regulated genes in pro‐B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro‐B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro‐B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B‐cell identity and function by regulating distinct target genes in early and late B lymphopoiesis. Genome‐wide sequencing approaches reveal that the transcription factor Pax5 controls the identity and function of B cells by regulating the expression of distinct target genes in pro‐B and mature B cells.

BackgroundB/T cell aggregates in the salivary glands (SG) of Sjögren's syndrome (SS) can give rise to ectopic lymphoid structures (ELS) forming ectopic germinal centers (GCs), which has been linked to the development of MALT lymphoma (MALT-L). T follicular-helper cells (Tfh) and T follicular-regulatory cells (Tfr) are specialized CD4+ T-cells that positively and negatively regulate, respectively, the magnitude of the GCs response and the onset of autoimmunity.ObjectivesTo characterize the infiltration of Tfh and Tfr in the SG infiltrates of patients with SS in the context of the presence/absence of ectopic GCs and in subjects with MALT-L.MethodsSG biopsies with matching histology and RNA from 37 SS and 38 non-specific chronic sialadenitis (NSCS) patients were stratified as ELS-/ELS+ based on CD3/CD20/CD21/CD138 immunostaining (IHC). Histological samples and mRNA from 12 parotid MALT-L were also studied. Gene expression was measured by Taqman rt-PCR. Multicolor immunofluorescence/confocal microscopy for CD3, CD4, CD45RO, ICOS, PD1, BCL6 and FoxP3 was used to identify Tfh and Tfr.ResultsTfh (CD4+CD45RO+PD1+ICOS+FoxP3-) cells and Tfr (CD4+CD45RO+PD1+ICOS+FoxP3+) cells were strongly enriched in ELS+ vs ELS- SS samples. The Tfh:Tfr ratio in ELS+ SG was approximately 2:1. Interestingly, while in tonsils Tfr were routinely detected within GCs, in ELS+ SG Tfr were predominantly excluded from the B cell follicles and accumulated in the T cell rich areas at the periphery of the lymphoid aggregates. Conversely, Tfh densely infiltrated the B cell rich areas and, within ectopic GCs, acquired BCL6. Furthermore, Tfh infiltration closely correlated with SG IL-21 mRNA expression, which in turn was strongly correlated with CD3, CD20 and CD138 IHC scores and with CXCL13, LTb, BAFF, AID and Pax5 gene expression. Finally, MALT-L samples displayed 10-fold higher IL-21 mRNA and twice as much PD1+ICOS+BCL6+ Tfh-cells/field compared to ELS+ SS samples.ConclusionsWithin the SG of SS patients Tfh cells closely segregate with lesional IL-21 expression, localize within ELS and are strongly enriched during MALT-L development. Conversely, although Tfr cells are also recruited to ELS+ SG in SS patients, we consistently demonstrated follicular exclusion of this subset from ectopic GCs. This suggests that Tfr in SS SG fail to exert their physiological immunoregulatory properties in controlling the magnitude of the GCs response and B cell autoreactivity, as observed in tonsils.AcknowledgementsThis work was supported by project grants from the Medical Research Council (MR/N003063/1 to MB) and Arthritis Research UK (grant 20089 to MB).Disclosure of InterestE. Pontarini: None declared, W. Murray-Brown: None declared, C. Croia: None declared, E. Astorri: None declared, D. Lucchesi: None declared, N. Lepse: None declared, N. Sutcliffe: None declared, A. Tappuni: None declared, C. Pitzalis: None declared, M. Bombardieri Consultant for: Amgen/Medimmune, GSK and UCB

Acute lymphoblastic leukemia (ALL) is the most common cancer during childhood, representing about 30–35% of cases. Its etiology is complex and not fully understood. ALL is influenced by genetic variants, and their frequencies (Fq) vary in different ethnic groups, which consequently could influence the epidemiology of this cancer worldwide. The aim of this study was to investigate the correlation between the genetic variants and their impacts on incidence (IC), mortality (MT), and prevalence (PV) rates of ALL in different world populations. Methods: Sixty variants were selected from the literature with Genome Wide Association studies (GWAS). Information regarding allele Fq was selected from the 1000 Genomes platform. Epidemiological data were taken from the Global Burden of disease visualisations (GBD) Compare website. Statistical analyses were calculated in RStudio v.3.5.1 software. Results: Four variants demonstrated significant results in correlations with epidemiological data for ALL. The PAX5 gene variant (rs2297105) had an indirect relationship with PV and IC of ALL, showing that an increased Fq of this variant is related to low rates of both. An increased Fq of rs915172 in EPB4IL2 gene was also correlated with a lower IC of ALL. The rs1048943 of the CYP1A1 gene and the rs3088440 polymorphism of the CDKN2A gene were shown to have a direct proportional relationship with MT rate, showing that an increased Fq of these variants correlates with a worse prognosis worldwide. Conclusion: Our study points out four important variants for understanding the IC, PV, and MT rates for ALL. The ascertainment of these data may help to choose molecular markers to investigate the susceptibility and prognosis of ALL.

Background The use of high-throughput analytical techniques has enabled the description of acute lymphoblastic leukaemia (ALL) subtypes. The TCF3-HLF translocation is a very rare rearrangement in ALL that is associated with an extremely poor prognosis. The TCF3-HLF fusion gene in the described case resulted in the fusion of the homeobox-related gene of TCF3 to the leucine zipper domain of HLF . The TCF3-HLF fusion gene product acts as a transcriptional factor leading to the dedifferentiation of mature B lymphocytes into an immature state (lymphoid stem cells). This process initiates the formation of pre-leukaemic cells. Due to the rarity of this chromosomal aberration, only a few cases have been described in the literature. The advantage of this work is the presentation of an interesting case of clonal evolution of cancer cells and the cumulative implications (diagnostic and prognostic) of the patient’s genetic alterations. Case presentation This work presents a patient with diagnosed with TCF3-HLF -positive ALL. Moreover, the additional genetic alterations, which play a key role in the pathogenesis of ALL, were detected in this patient: deletion of a fragment from the long arm of chromosome 13 (13q12.2-q21.1) containing the RB1 gene, intragenic deletions within the PAX5 gene and NOTCH1 intragenic duplication. Conclusions A patient with coexistence of chromosomal alterations and the TCF3-HLF fusion has not yet been described. Identifying all these chromosomal aberrations at the time of diagnosis could be sufficient to determine the cumulative effects of the described deletions on the activity of other oncogenes or tumour suppressors, as well as on the clinical course of the disease. On the other hand, complex changes in the patient’s karyotype and clonal evolution of cancer cells call into question the effectiveness of experimental therapy.