Committed Human CD23-Negative Light-Zone Germinal Center B Cells Delineate Transcriptional Program Supporting Plasma Cell Differentiation

B cell affinity maturation occurs in the germinal center (GC). Light-zone (LZ) GC B cells (BGC-cells) interact with follicular dendritic cells (FDCs) and compete for the limited, sequential help from T follicular helper cells needed to escape from apoptosis and complete their differentiation. The highest-affinity LZ BGC-cells enter the cell cycle and differentiate into PCs, following a dramatic epigenetic reorganization that induces transcriptome changes in general and the expression of the PRDM1 gene in particular. Human PC precursors are characterized by the loss of IL-4/STAT6 signaling and the absence of CD23 expression. Here, we studied the fate of human LZ BGC-cells as a function of their CD23 expression. We first showed that CD23 expression was restricted to the GC LZ, where it was primarily expressed by FDCs; less than 10% of tonsil LZ BGC-cells were positive. Sorted LZ BGC-cells left in culture and stimulated upregulated CD23 expression but were unable to differentiate into PCs – in contrast to cells that did not upregulate CD23 expression. An in-depth analysis (including single-cell gene expression) showed that stimulated CD23-negative LZ BGC-cells differentiated into plasmablasts and time course of gene expression changes delineates the transcriptional program that sustains PC differentiation. In particular, we identified a B cell proliferation signature supported by a transient MYC gene expression. Overall, the CD23 marker might be of value in answering questions about the differentiation of normal BGC-cells and allowed us to propose an instructive LZ BGC-cells maturation and fate model.

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The t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile

Blood ◽

10.1182/blood.v99.7.2285 ◽

2002 ◽

Vol 99 (7) ◽

pp. 2285-2290 ◽

Cited By ~ 202

Author(s):

James Z. Huang ◽

Warren G. Sanger ◽

Timothy C. Greiner ◽

Louis M. Staudt ◽

Dennis D. Weisenburger ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Gene Expression Profile ◽

Expression Profile ◽

Germinal Center ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Germinal Center B Cell ◽

Cell Gene Expression ◽

Cell Gene

Recently we have identified subgroups of de novo primary diffuse large B-cell lymphoma (DLBCL) based on complementary DNA microarray-generated gene expression profiles. To correlate the gene expression profiles with cytogenetic abnormalities in these DLBCLs, we examined the occurrence of the t(14;18)(q32;q21) in the 2 distinctive subgroups of DLBCL: one with the germinal center B-cell gene expression signature and the other with the activated B cell–like gene expression signature. The t(14;18) was detected in 7 of 35 cases (20%). All 7 t(14;18)-positive cases had a germinal center B-cell gene expression profile, representing 35% of the cases in this subgroup, and 6 of these 7 cases had very similar gene expression profiles. The expression of bcl-2 and bcl-6 proteins was not significantly different between the t(14;18)-positive and -negative cases, whereas CD10 was detected only in the group with the germinal center B-cell expression profile, and CD10 was most frequently expressed in the t(14;18)-positive cases. This study supports the validity of subdividing DLBCL into 2 major subgroups by gene expression profiling, with the t(14;18) being an important event in the pathogenesis of a subset of DLBCL arising from germinal center B cells. CD10 protein expression is useful in identifying cases of DLBCL with a germinal center B-cell gene expression profile and is often expressed in cases with the t(14;18).

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Cbl and Cbl-b control the germinal center reaction by facilitating naive B cell antigen processing

Journal of Experimental Medicine ◽

10.1084/jem.20191537 ◽

2020 ◽

Vol 217 (9) ◽

Cited By ~ 1

Author(s):

Xin Li ◽

Liying Gong ◽

Alexandre P. Meli ◽

Danielle Karo-Atar ◽

Weili Sun ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Antigen Processing ◽

Cell Interaction ◽

Affinity Maturation ◽

Antigen Uptake ◽

Cell Antigen ◽

Germinal Center Reaction ◽

Follicular Helper

Antigen uptake and presentation by naive and germinal center (GC) B cells are different, with the former expressing even low-affinity BCRs efficiently capture and present sufficient antigen to T cells, whereas the latter do so more efficiently after acquiring high-affinity BCRs. We show here that antigen uptake and processing by naive but not GC B cells depend on Cbl and Cbl-b (Cbls), which consequently control naive B and cognate T follicular helper (Tfh) cell interaction and initiation of the GC reaction. Cbls mediate CD79A and CD79B ubiquitination, which is required for BCR-mediated antigen endocytosis and postendocytic sorting to lysosomes, respectively. Blockade of CD79A or CD79B ubiquitination or Cbls ligase activity is sufficient to impede BCR-mediated antigen processing and GC development. Thus, Cbls act at the entry checkpoint of the GC reaction by promoting naive B cell antigen presentation. This regulation may facilitate recruitment of naive B cells with a low-affinity BCR into GCs to initiate the process of affinity maturation.

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Activation Induced Cytidine Deaminase (AID) Is Required for Germinal-Center Derived Lymphomagenesis.

Blood ◽

10.1182/blood.v108.11.223.223 ◽

2006 ◽

Vol 108 (11) ◽

pp. 223-223

Author(s):

Laura Pasqualucci ◽

Mara Compagno ◽

Tongwei Mo ◽

Paula Smith ◽

Herbert C. Morse ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Somatic Hypermutation ◽

Profile Analysis ◽

Cytidine Deaminase ◽

Receptor Gene ◽

Activation Induced Cytidine Deaminase ◽

Hodgkin's Lymphomas

Abstract Most B cell non-Hodgkin’s lymphomas (B-NHL) derive from germinal center (GC) B cells and their pathogenesis is associated with the accumulation of distinct genetic lesions, including chromosomal translocations and a more recently identified mechanism of genomic instability, termed aberrant somatic hypermutation. These alterations are thought to be due to mistakes occurring during two GC-associated immunoglobulin (Ig) genes remodeling processes: class switch recombination (CSR) and somatic hypermutation (SHM). However, this model has never been formally proven. To conclusively investigate the role of CSR and SHM in the pathogenesis of B-NHL, we examined whether lymphoma development in mice requires the function of activation induced cytidine deaminase (AID), a DNA editing enzyme expressed specifically in GC and activated B cells and essential for both processes. Three transgenic mouse models were generated by crossing lymphoma-prone mice (λMYC, λMYC/IμHABCL6 and IμHABCL6) with mice (AID−/−) that are unable to undergo both SHM and CSR. The λMYC mice develop a diffusely infiltrating monoclonal proliferation of pre-GC origin, with unmutated IgV genes and lack of BCL6 expression, and therefore presumably independent from AID-associated DNA remodeling events. Conversely, lymphomas in λMYC/IμHABCL6 and IμHABCL6 mice recapitulate GC/post GC-derived malignancies, in that the former display somatically mutated IgV genes and upregulation of post-GC markers (CD138) in most of the cases, while the latter develop a splenic lymphoproliferative syndrome that culminates, past 12 months of age, in clonal B cell lymphomas with DLBCL morphology and somatically mutated IgV genes (~70% of the animals) (Cattoretti et al., Cancer Cell 7:445–455, 2005). Mice were monitored for tumor incidence and survival, and a combination of histologic, immunophenotypic and gene expression profiling analysis was used for tumor characterization. As expected, no significant differences in event-free survival and lymphoma type were observed between AID-proficient and AID-deficient λMYC mice, in agreement with their pre-GC derivation. Conversely, a phenotypic shift of the tumor was observed in λMYC/IμHABCL6 mice when bred into an AID−/− background, with >80% of the cases (N=21/26) reverting to a pre-GC phenotype (loss of GC/post GC markers) undistinguishable from that of the λMYC and λMYC/AID−/− mice. Gene expression profile analysis on representative cases (N=10 λMYC/IμHABCL6 and 5 each for λMYC, λMYC/AIDKO, λMYC/IμHABCL6/AIDKO) confirmed significant phenotypic similarities between pre-GC derived λMYC lymphomas and the λMYC/IμHABCL6/AID −/− lymphomas, which co-segregated in a separate cluster from λMYC/IμHABCL6 tumors. Analogously, a significant reduction in DLBCL frequency was observed in the IμHABCL6/AIDKO cohort as compared to IμHABCL6 mice (N= 4/19, 21% vs 8/14, 57%; p=0.03). Taken together, these results indicate that GC-derived lymphomas cannot develop in the absence of AID, thereby providing direct support to the notion that AID-mediated mistakes in antigen receptor gene modification events (CSR and SHM) represent major contributors to B-NHL pathogenesis.

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DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Blood ◽

10.1182/blood.v110.11.685.685 ◽

2007 ◽

Vol 110 (11) ◽

pp. 685-685 ◽

Cited By ~ 1

Author(s):

Rita Shaknovich ◽

Leandro Cerchietti ◽

Maria E. Figueroa ◽

Ari Melnick

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Dna Methyltransferase ◽

Critical Role ◽

Epigenetic Programming ◽

Differential Digestion ◽

Epigenetic Signatures

Abstract Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

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WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.646077 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yanan Li ◽

Anshuman Bhanja ◽

Arpita Upadhyaya ◽

Xiaodong Zhao ◽

Wenxia Song

Keyword(s):

B Cells ◽

B Cell ◽

Lipid Bilayers ◽

Germinal Center ◽

Actin Polymerization ◽

Immunological Synapse ◽

Somatic Hypermutation ◽

Affinity Maturation ◽

Membrane Protrusions ◽

Germinal Center B Cells

B-cells undergo somatic hypermutation and affinity maturation in germinal centers. Somatic hypermutated germinal center B-cells (GCBs) compete to engage with and capture antigens on follicular dendritic cells. Recent studies show that when encountering membrane antigens, GCBs generate actin-rich pod-like structures with B-cell receptor (BCR) microclusters to facilitate affinity discrimination. While deficiencies in actin regulators, including the Wiskott-Aldrich syndrome protein (WASp), cause B-cell affinity maturation defects, the mechanism by which actin regulates BCR signaling in GBCs is not fully understood. Using WASp knockout (WKO) mice that express Lifeact-GFP and live-cell total internal reflection fluorescence imaging, this study examined the role of WASp-mediated branched actin polymerization in the GCB immunological synapse. After rapid spreading on antigen-coated planar lipid bilayers, GCBs formed microclusters of phosphorylated BCRs and proximal signaling molecules at the center and the outer edge of the contact zone. The centralized signaling clusters localized at actin-rich GCB membrane protrusions. WKO reduced the centralized micro-signaling clusters by decreasing the number and stability of F-actin foci supporting GCB membrane protrusions. The actin structures that support the spreading membrane also appeared less frequently and regularly in WKO than in WT GCBs, which led to reductions in both the level and rate of GCB spreading and antigen gathering. Our results reveal essential roles for WASp in the generation and maintenance of unique structures for GCB immunological synapses.

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Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells

10.1101/2020.11.17.385716 ◽

2020 ◽

Author(s):

Juhee Pae ◽

Jonatan Ersching ◽

Tiago B. R. Castro ◽

Marta Schips ◽

Luka Mesin ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Clonal Expansion ◽

Affinity Maturation ◽

Cyclin D3 ◽

Dark Zone ◽

Follicular Helper ◽

Cell Cycle Regulator Cyclin

AbstractDuring affinity maturation, germinal center (GC) B cells alternate between proliferation and so-matic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively-selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, to clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.

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Brg1 Supports B Cell Proliferation and Germinal Center Formation Through Enhancer Activation

Frontiers in Immunology ◽

10.3389/fimmu.2021.705848 ◽

2021 ◽

Vol 12 ◽

Author(s):

Dominik Schmiedel ◽

Hadas Hezroni ◽

Amit Hamburg ◽

Ziv Shulman

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Transcriptional Activation ◽

Germinal Center ◽

Cell Activation ◽

Critical Role ◽

B Cell Activation ◽

Germinal Center Formation

Activation and differentiation of B cells depend on extensive rewiring of gene expression networks through changes in chromatin structure and accessibility. The chromatin remodeling complex BAF with its catalytic subunit Brg1 was previously identified as an essential regulator of early B cell development, however, how Brg1 orchestrates gene expression during mature B cell activation is less clear. Here, we find that Brg1 is required for B cell proliferation and germinal center formation through selective interactions with enhancers. Brg1 recruitment to enhancers following B cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle-associated genes. Accordingly, Brg1-deficient B cells were unable to mount germinal center reactions and support the formation of class-switched plasma cells. Our findings show that changes in B cell transcriptomes that support B cell proliferation and GC formation depend on enhancer activation by Brg1. Thus, the BAF complex plays a critical role during the onset of the humoral immune response.

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Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

Journal of Experimental Medicine ◽

10.1084/jem.20171450 ◽

2018 ◽

Vol 215 (6) ◽

pp. 1571-1588 ◽

Cited By ~ 70

Author(s):

Norbert Pardi ◽

Michael J. Hogan ◽

Martin S. Naradikian ◽

Kaela Parkhouse ◽

Derek W. Cain ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Neutralizing Antibodies ◽

Affinity Maturation ◽

Modified Nucleosides ◽

Vaccine Platform ◽

Intradermal Vaccination ◽

Cell Responses ◽

Follicular Helper

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

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Recurrent 11q24.3 Gain Contributes to the Pathogenesis of Diffuse Large B Cell Lymphoma (DLBCL) by Deregulating ETS1 and FLI1

Blood ◽

10.1182/blood.v118.21.1386.1386 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1386-1386

Author(s):

Paola Bonetti ◽

Monica Testoni ◽

Marta Scandurra ◽

Maurilio Ponzoni ◽

Roberto Piva ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

B Cell ◽

Germinal Center ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Functional Characterization ◽

B Cell Lymphoma ◽

Transcriptional Program ◽

Down Regulation

Abstract Abstract 1386 DLBCL represents the most common form of B-cell non-Hodgkin lymphoma (B-NHL). It is an aggressive and heterogeneous disease, comprising at least three distinct subtypes based on gene expression profile analysis: germinal center B cell-like DLBCL (GCB), activated B cell-like DLBCL (ABC) and primary mediastinal B-cell lymphoma (PMBL). These subtypes are supposed to derive from B cells at different stages of differentiation. Normal germinal center (GC) B-cell differentiation requires a complex transcriptional program and alterations of genes involved in this process are relevant for DLBCL pathogenesis. Identification and functional characterization of new genetic lesions would provide critical information to better understand the pathogenesis of DLBCL. With this aim, we studied the genomic profiles of 166 DLBCL patients, identified and characterized a recurrent gain mapping to chromosome 11q24.3. Methods. Genomic profiles were obtained from 166 Affymetrix 250K SNP arrays and integrated with gene expression data (GeneChip U133 plus 2.0) in 54 cases. Data were validated by PCR and immunohistochemistry. Gene silencing experiments were done with shRNA. Results. A minimal common region 11q24.3 gain was present in 26% of DLBCL samples and it encompassed six genes (ETS1, FLI1, KCNJ1, KCNJ5, P53AIP1, RICS). Samples with the 11q24.3 gain were significantly associated with high expression of the transcription factors ETS1 and FLI1. Data were confirmed by real-time PCR and by immunohistochemical analysis. Gene expression analysis showed 228 transcripts with a significantly different expression between cases with or without the lesion (p<0.01, >2-fold change): 215 genes were up-regulated in the patients with the gain and 13 were down-regulated, suggesting that this lesion has an impact on the transcriptional program of the tumor cells. To study the biological meaning of the lesion, ETS1 and FLI1 expression was down-regulated in a DLBCL cell line bearing the same lesion observed in clinical specimens (OCI-Ly7). Results showed that ETS1 and FLI1 down-regulation caused a reduced proliferation rate and activation of apoptosis leading to cell death. Concomitant ETS1 and FLI1 down-regulation resulted in a more severe phenotype. Only FLI1 was confirmed to be essential for cell viability in other DLBCL cell lines (SUDHL4, VAL, U2932), whereas ETS1 did not, suggesting a distinct role of the two transcription factors in different DLBCL samples. Preliminary results showed that down-regulation of ETS1 affected the transcriptional program of GC B-cell terminal differentiation causing an up-regulation of BLIMP1, the master regulator of plasma cells differentiation. Conclusions. In DLBCL, a recurrent gain at 11q24.3 determines the over-expression of the transcription factors ETS1 and FLI1. Functional experiments showed that the lesion might sustain DLBCL proliferation and viability, and contribute to a differentiation blockade of the GC B-cell towards a plasma cell lineage by negatively regulating BLIMP1. Disclosures: No relevant conflicts of interest to declare.

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Three-Dimensional Reorganization of the Genome During B Cell Affinity Maturation

Blood ◽

10.1182/blood.v120.21.279.279 ◽

2012 ◽

Vol 120 (21) ◽

pp. 279-279

Author(s):

Karen L. Bunting ◽

David Soong ◽

Yanwen Jiang ◽

Brandon L. Swed ◽

Katerina Hatzi ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Three Dimensional ◽

Affinity Maturation ◽

Cell Maturation ◽

Transcriptional Reprogramming ◽

B Cell Maturation ◽

Genome Wide ◽

Cell Affinity

Abstract Abstract 279 B cell affinity maturation is fundamental to the development of humoral immunity. To create a diverse antibody repertoire, B cells activated in the germinal centre (GC) must undergo a profound change in phenotype. This unique phenotypic change, which features simultaneous proliferation and somatic hypermutation and which can predispose to the development of lymphoma, requires radically altered gene expression programming in GC B cells. However, the way that this gene expression program is coordinated is unknown. Emerging evidence suggests that the higher-order organization of chromatin plays a role in the co-regulation of genes. We hypothesised that the three-dimensional organization of genes and chromosomes in the nucleus of B cells plays a key role in the epigenetic and transcriptional reprogramming that underlies acquisition of the GC B cell phenotype during B cell maturation. Using genome-wide mapping of chromatin interactions (Hi-C), combined with genome-wide profiles of gene expression (RNA-seq), histone modifications and transcription factor binding (ChIP-seq) in human naïve B (NB) and GC B cells, we have discovered that the three-dimensional structure of the genome undergoes widespread reorganization during B cell maturation to coordinate the GC transcriptional programme. Conformational maps of chromosome folding in these cells reveal a novel and profound loss of inter-arm interactions, reflecting lower chromosome compaction in GC B cells. Remarkably, we observed extensive differential partitioning of genes into NB- and GC B cell-specific compartments, and demonstrate for the first time that coordinated changes in histone modifications (H3K4Me2: P=3×10−35; H3K27Ac: P=3×10−33; Fisher's exact test) and transcription (P=1×10−9) required for cell type specification is mediated by the de novo formation of precisely delimited chromosome neighbourhoods. Most strikingly, we find that remodelling of the GC B cell genome involves the specific structural unlocking of genes that drive the GC transcriptional programme, such as AICDA, MTA3, and BCL6. Coordinate activation of these genes is mediated by the expansion of gene interaction neighbourhoods, increased promoter interactivity (P=3×10−35), engagement of long-range enhancer-promoter interactions (>2-fold increase), and the formation of gene body loops (P=3.18×10−15). Intriguingly, the master regulator of GC B cell differentiation, BCL6, shows a high propensity for all of these different types of interactions, suggesting that regulation of this gene in the context of chromatin is highly complex. Integration with genome-wide binding data for the structural organizing proteins, CTCF and cohesin, as well as the cell-specific factor, PU.1, supports a specific role for these proteins in the repositioning of activated promoters and enhancer regions during B cell maturation. This study shows for the first time that the architecture of the genome is critical for specification of cellular phenotype, and that epigenetic and transcriptional reprogramming in GC B cells is functionally linked to the structural reorganization of genes in the nucleus. Importantly, the higher-order organization of chromatin could represent a novel mechanism by which GC B cell gene expression is dysregulated in lymphoma. Disclosures: No relevant conflicts of interest to declare.

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