Distinct roles for E12 and E47 in B cell specification and the sequential rearrangement of immunoglobulin light chain loci

The E2A gene products, E12 and E47, are critical regulators of B cell development. However, it remains elusive whether E12 and E47 have overlapping and/or distinct functions during B lymphopoiesis. We have generated mice deficient for either E12 or E47 and examined their roles in B cell maturation. We show that E47 is essential for developmental progression at the prepro–B cell stage, whereas E12 is dispensable for early B cell development, commitment, and maintenance. In contrast, both E12 and E47 play critical roles in pre–B and immature B cells to promote immunoglobulin λ (Igλ) germline transcription as well as Igλ VJ gene rearrangement. Furthermore, we show that E12 as well as E47 is required to promote receptor editing upon exposure to self-antigen. We demonstrate that increasing levels of E12 and E47 act to induce Igλ germline transcription, promote trimethylated lysine 4 on histone 3 (H3) as well as H3 acetylation across the Jλ region, and activate Igλ VJ gene rearrangement. We propose that in the pre–B and immature B cell compartments, gradients of E12 and E47 activities are established to mechanistically regulate the sequential rearrangement of the Ig light chain genes

ELUCIDATING REGULATORY NETWORKS PROMOTING B-CELL DEVELOPMENT

B-cells are an essential part of our adaptive immune system. A network of transcription factors together with external signals facilitate the gradual developmental progression from progenitor cells towards the B-cell fate. Several key factors participating in this network have been identified. Among these are the transcription factors E2A, HEB, EBF1 and FOXO1 as well as the IL7 signaling cascade. The objective of this thesis has been to increase our understanding of the transcriptional network orchestrating B-lineage specification and commitment. Based on the combined expression of surface markers and transgenic reporter genes, we have identified three hierarchically related and functionally distinct subpopulations within the common lymphoid progenitor (CLP) compartment. Using this knowledge, we have re-evaluated previously characterized knock-out mouse models in order to obtain a higher resolution analysis of critical events in early B-cell commitment. Based on these studies, we propose a transcriptional hierarchy where the transcription factors E2A and HEB initiate the B-cell specification program in the LY6D- CLPs through up regulation of FOXO1. During the transition to LY6D expressing CLPs, E2A and FOXO1 induce EBF1. Subsequently, FOXO1 and EBF1 generate a feed-forward loop, leading to activation of PAX5, B-cell commitment and the progression to the CD19+ pro-B cell stage

Regulation of gene expression in the immune system and in virally-transformed cells

The correct development and functioning of the immune system is critical for the defence of the host organism against pathogens and cancers. V(D)J recombination generates diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes by the regulated joining of variable (V), diversity (D) and joining (J) gene segments. Tissue-specific enhancers in the DNA genome activate these genes to undergo recombina-tion by triggering non-coding transcription through the recombining gene segments, following interaction with the respective promoters. How this is achieved is un-known. The specificity of enhancer/promoter interactions was examined using the murine Igλ chain locus. The transcription factors that bind to the three main promoters were identified by DNase I footprinting. Of these, a factor termed E47 was shown to interact with IRF4 by co-immunoprecipitation experiments. The importance of these interactions was confirmed by mutagenesis where it was shown that mutations of any of the binding sites in DNA for the transcription factors or mutations in the amino acids involved in protein-protein interactions decreased the rate of transcrip-tion. Together, these studies suggest that IRF4/E47 interactions may play a key role in triggering locus activation. RNA-Seq data from HPV-positive samples and cell lines were analysed to identify putative biomarkers for cervical cancer. Infection with HPVs is the main cause for cervical cancer accounting for 10-15% of cancer-related deaths in women world-wide. It is established that HPVs escape the immune response over decades to es-tablish tumorigenesis but the specific mechanism is unknown. Virus integration into the host genome and deregulation of several genes may play a key role in promot-ing cancer; of particular interest are those transcripts that form the “surfacesome”. Among these, particular interest was given to connexin 26 (Cx26), which is classified as cancer-predisposition gene and was found to be commonly down regulated in all samples analysed. Recombinant adenoviruses expressing the two HPV16 oncogenes were generated and employed to transduce HaCaT cells to analyse Cx26 mRNA and protein levels coupled with dye transfer assays to study the structural behaviour of connexins. The data presented showed that E6 and E7 alter Cx26 protein expression by relocating Cx26 within the cytoplasm from the membrane-bound form. This was confirmed in the dye transfer assay where cell-cell communications were los

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR.Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells.This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.</p

Regulation of B Cell Gene Expression and Function by Ikaros, Helios and Bcl6

B lymphocytes constitute a key branch of adaptive immunity by providing specificity to recognize a vast variety of antigens by B cell antigen receptors (BCR) and secreted antibodies. Antigen recognition activates the cells and can produce antibody secreting plasma cells via germinal center reaction that leads to the maturation of antigen recognition affinity and switching of antibody effector class. The specificity of antigen recognition is achieved through a multistep developmental pathway that is organized by interplay of transcription factors and signals through BCR. Lymphoid malignancies arise from different stages of development in abnormal function of transcriptional regulation. To understand the B cell development and the function of B cells, a thorough understanding of the regulation of gene expression is important. The transcription factors of the Ikaros family and Bcl6 are frequently associated with lymphoma generation. The aim of this study was to reveal the targets of Ikaros, Helios and Bcl6 mediated gene regulation and to find out the function of Ikaros and Helios in B cells. This study uses gene targeted DT40 B cell lines and establishes a role for Ikaros family factors Ikaros and Helios in the regulation of BCR signaling that is important at developmental checkpoints, for cell survival and in activation. Ikaros and Helios had opposing roles in the regulation of BCR signals. Ikaros was found to directly repress the SHIP gene that encodes a signaling lipid-metabolizing enzyme, whereas Helios had activating effect on SHIP expression. The findings demonstrate a balancing function for these two Ikaros family transcription factors in the regulation of BCR signaling as well as in the regulation of gene expression. Bcl6 was found to repress plasma cell gene expression program while maintaining gene expression profile of B cells. Analysis of direct Bcl6 target genes suggested novel mechanisms for Bcl6-mediated suppression of plasma cell differentiation and promoting germinal center phenotype.Siirretty Doriast

Formation of the Immunoglobulin Repertoire in Precursor-B-cell Development

Precursor-B cells develop in bone marrow (BM) from hematopoietic stem cells (HSC) with the ultimate goal to generate mature B-cells with unique immunoglobulins (Ig), whereby all B cells together provide an enormous Ig repertoire diversity. Formation of the Ig occurs through somatic V(D)J recombination of the Ig heavy chain (IGH) and the Ig light chain (IGK or IGL) loci that contain multiple variable (V), diverse (D) and joining (J) coding elements. The Ig loci recombine in a ordered manner. The IGH locus rearranges before the Ig light chain loci and functional Ig rearrangements are generally restricted to one allele. Thus, the ordered accessibility of Ig loci for rearrangements is tightly controlled. Studies described in this thesis aimed to better understand the mechanisms underlying the stage-specific regulation of V(D) J recombination and the generation of a diverse Ig repertoire during precursor-B-cell development. The stepwise V(D)J recombination process is controlled by transcriptional and epigenetic mechanisms which must cooperate together to provide the developmental increase in Ig loci accessibility for rearrangement. The formation of the broad Ig repertoire is dependent on the B-cell developmental niche. The BM niche undergoes physiological changes during life and therefore provides different signals to developing B cells, resulting in distinct Ig gene repertoires with distinct reactivity. Future studies should reveal which environmental factors modify the Ig loci accessibility for the V(D)J recombinase and thereby control the Ig repertoire and reactivity. Such knowledge might be applicable for shaping the Ig repertoire in immune deficient patients

Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles

Globin Gene Expression: Role of Transcription Factors

La dérégulation de l'expression génétique est une base pathophysiologique de plusieurs maladies. On a utilisé le locus du gène β-globine humain comme modèle pour élucider le mécanisme de régulation de la transcription génétique et évaluer son expression génétique durant l'érythropoïèse. La famille des protéines 'E' est composée de facteurs de transcription qui possèdent plusieurs sites de liaison au sein de locus du gène β-globine, suggérant leur rôle potentiel dans la régulation de l’expression de ces gènes. Nous avons montré que les facteurs HEB, E2A et ETO2 interagissent d’une manière significative avec la région contrôle du Locus (LCR) et avec les promoteurs des gènes de la famille β-globine. Le recrutement de ces facteurs au locus est modifié lors de l'érythropoïèse dans les cellules souches hematopoitiques et les cellules erythroides de souris transgéniques pour le locus de la β-globine humain, ainsi que dans les cellules progénitrices hématopoïétiques humaines. De plus par cette étude, nous démontrons pour la première fois que le gène β-globine humain est dans une chromatine active et qu’il interagit avec des facteurs de transcriptions de type suppresseurs dans les cellules progénitrices lymphoïdes (voie de différentiation alternative). Cette étude a aussi été faite dans des souris ayant une génétique mutante caractérisée par l'absence des facteurs de transcription E2A ou HEB.Aberrant gene expression is an underlying pathophysiology in many disease conditions. Lineage-specification and -commitment is tightly dependent on lineage-specific transcription factors to regulate the expression of target genes. Using human β-globin locus as a model, we investigated how the transcriptional machinery is set and regulated during erythropoiesis and how it impacts globally on gene expression. Class I bHLH proteins are important transcription factors whose binding sites are frequently clustered throughout the β-globin gene locus, suggesting their role in globin gene regulation. We showed that, in hematopoietic progenitor (HPC) and erythroid cells (EryC) of the transgenic mouse for human β-globin locus and human HPC cells (CD34+); HEB, E2A and ETO-2 significantly interact with locus control region (LCR) and promoters of globin genes, and their relative ratio is altered during erythropoiesis. For the first time, we found that in other hematopoietic lineages, human β-globin locus is in active chromatin and interacts with transcription factors involved in repression. Strikingly and consistent with the expression of globin genes, we characterized transcription factors involved in open chromatin configuration and basal level of globin gene expression in lymphoid progenitor cells. Further, with the genetic power of E2A and HEB knockout mice, our findings were clarified in mutant backgrounds