Rôle des éléments cis-régulateurs du locus des chaines lourdes d’immunoglobuline sur l’organisation du noyau et l’intégrité du génome du lymphocyte B

The late stages of B lymphocyte differentiation are characterized by gene rearrangements, such as somatic hypermutation and class recombination, which ultimately lead to the production of highly specific immunoglobulins. These processes, occurring at immunoglobulin-coding loci, introduce mutations and DNA double-strand breaks. Any malfunctioning of these rearrangements constitutes a major threat to the integrity of the B cell genome, and their regulation is therefore essential. Monitoring and control of these events are mediated by the cis-regulatory regions of the IgH locus: the EµMARs and 3'RR regions. The role of these regions at transcriptional level has been extensively characterized, using various mouse models with partial or total deletions of these regions. The spatio-temporal regulation of gene rearrangements has also been described at the nuclear level, with dynamic shifts of immunoglobulin loci, as well as at the supranucleosomal level through the formation of chromatin loops. Data from the laboratory and the literature suggest the involvement of cis-regulatory regions of the IgH locus in these additional regulatory mechanisms. We have demonstrated that core enhancers of the 3'RR are necessary and sufficient to correctly address the IgH locus in the nucleus and to allow chromatin loop formation and thus induce optimal and functional CSR recombination. However, our data indicate that these core enhancers are not sufficient to maintain the genomic integrity of the B cell, as shown by the analysis of mutations in oncogenes such as Bcl6. This work opens up new perspectives for understanding the mechanisms governing late B development and the maintenance of genomic integrity via nuclear organization.Les stades tardifs de la différenciation lymphocytaire B sont caractérisés par des remaniements géniques, telles que l’hypermutation somatique et la recombinaison de classe, qui in fine, permettent la production d’immunoglobulines hautement spécifique. Ces processus, intervenant aux loci codant les immunoglobulines, introduisent des mutations ainsi que des cassures double brin de l’ADN. Tout dysfonctionnement de ces remaniements constitue un danger majeur pour l’intégrité du génome de la cellule B ; leur régulation est donc essentielle. La surveillance et le contrôle de ces évènements sont notamment médiés par les régions cis-régulatrices du locus IgH : les régions EµMARs et 3’RR. Le rôle de ces régions à l’échelle transcriptionnelle a été largement caractérisé, grâce à différents modèles murins présentant des délétions totales ou partielle de celles-ci. La régulation spatio-temporelle des remaniements géniques a été également décrite au niveau nucléaire, avec des déplacements dynamiques des loci d’immunoglobuline, ainsi qu’au niveau supranucléosomale par la formation de boucles de chromatine. Des données provenant du laboratoire et de la littérature suggèrent l’implication des régions cis-régulatrices du locus IgH dans ces mécanismes de régulation supplémentaires. Nous avons démontré que les core enhancers de la 3’RR sont nécessaires et suffisants pour correctement adresser le locus IgH dans le noyau et pour permettre la formation des boucles de chromatine et ainsi induire une recombinaison de CSR optimale et fonctionnelle. Toutefois, nos données indiquent que ces core enhancers ne sont pas suffisants pour maintenir l’intégrité génomique de la cellule B comme en témoigne l’analyse des mutations sur des oncogènes tels que Bcl6. Ces travaux ouvrent de nouvelles perspectives pour la compréhension des mécanismes régissant le développement B tardif ainsi que le maintien de l’intégrité génomique via l’organisation du noyau

Rôle des éléments cis-régulateurs du locus des chaines lourdes d’immunoglobuline sur l’organisation du noyau et l’intégrité du génome du lymphocyte B

The late stages of B lymphocyte differentiation are characterized by gene rearrangements, such as somatic hypermutation and class recombination, which ultimately lead to the production of highly specific immunoglobulins. These processes, occurring at immunoglobulin-coding loci, introduce mutations and DNA double-strand breaks. Any malfunctioning of these rearrangements constitutes a major threat to the integrity of the B cell genome, and their regulation is therefore essential. Monitoring and control of these events are mediated by the cis-regulatory regions of the IgH locus: the EµMARs and 3'RR regions. The role of these regions at transcriptional level has been extensively characterized, using various mouse models with partial or total deletions of these regions. The spatio-temporal regulation of gene rearrangements has also been described at the nuclear level, with dynamic shifts of immunoglobulin loci, as well as at the supranucleosomal level through the formation of chromatin loops. Data from the laboratory and the literature suggest the involvement of cis-regulatory regions of the IgH locus in these additional regulatory mechanisms. We have demonstrated that core enhancers of the 3'RR are necessary and sufficient to correctly address the IgH locus in the nucleus and to allow chromatin loop formation and thus induce optimal and functional CSR recombination. However, our data indicate that these core enhancers are not sufficient to maintain the genomic integrity of the B cell, as shown by the analysis of mutations in oncogenes such as Bcl6. This work opens up new perspectives for understanding the mechanisms governing late B development and the maintenance of genomic integrity via nuclear organization.Les stades tardifs de la différenciation lymphocytaire B sont caractérisés par des remaniements géniques, telles que l’hypermutation somatique et la recombinaison de classe, qui in fine, permettent la production d’immunoglobulines hautement spécifique. Ces processus, intervenant aux loci codant les immunoglobulines, introduisent des mutations ainsi que des cassures double brin de l’ADN. Tout dysfonctionnement de ces remaniements constitue un danger majeur pour l’intégrité du génome de la cellule B ; leur régulation est donc essentielle. La surveillance et le contrôle de ces évènements sont notamment médiés par les régions cis-régulatrices du locus IgH : les régions EµMARs et 3’RR. Le rôle de ces régions à l’échelle transcriptionnelle a été largement caractérisé, grâce à différents modèles murins présentant des délétions totales ou partielle de celles-ci. La régulation spatio-temporelle des remaniements géniques a été également décrite au niveau nucléaire, avec des déplacements dynamiques des loci d’immunoglobuline, ainsi qu’au niveau supranucléosomale par la formation de boucles de chromatine. Des données provenant du laboratoire et de la littérature suggèrent l’implication des régions cis-régulatrices du locus IgH dans ces mécanismes de régulation supplémentaires. Nous avons démontré que les core enhancers de la 3’RR sont nécessaires et suffisants pour correctement adresser le locus IgH dans le noyau et pour permettre la formation des boucles de chromatine et ainsi induire une recombinaison de CSR optimale et fonctionnelle. Toutefois, nos données indiquent que ces core enhancers ne sont pas suffisants pour maintenir l’intégrité génomique de la cellule B comme en témoigne l’analyse des mutations sur des oncogènes tels que Bcl6. Ces travaux ouvrent de nouvelles perspectives pour la compréhension des mécanismes régissant le développement B tardif ainsi que le maintien de l’intégrité génomique via l’organisation du noyau

Role of heavy chain locus cis-regulatory elements on nuclear organization and genome integrity of B cell

Les stades tardifs de la différenciation lymphocytaire B sont caractérisés par des remaniements géniques, telles que l’hypermutation somatique et la recombinaison de classe, qui in fine, permettent la production d’immunoglobulines hautement spécifique. Ces processus, intervenant aux loci codant les immunoglobulines, introduisent des mutations ainsi que des cassures double brin de l’ADN. Tout dysfonctionnement de ces remaniements constitue un danger majeur pour l’intégrité du génome de la cellule B ; leur régulation est donc essentielle. La surveillance et le contrôle de ces évènements sont notamment médiés par les régions cis-régulatrices du locus IgH : les régions EµMARs et 3’RR. Le rôle de ces régions à l’échelle transcriptionnelle a été largement caractérisé, grâce à différents modèles murins présentant des délétions totales ou partielle de celles-ci. La régulation spatio-temporelle des remaniements géniques a été également décrite au niveau nucléaire, avec des déplacements dynamiques des loci d’immunoglobuline, ainsi qu’au niveau supranucléosomale par la formation de boucles de chromatine. Des données provenant du laboratoire et de la littérature suggèrent l’implication des régions cis-régulatrices du locus IgH dans ces mécanismes de régulation supplémentaires. Nous avons démontré que les core enhancers de la 3’RR sont nécessaires et suffisants pour correctement adresser le locus IgH dans le noyau et pour permettre la formation des boucles de chromatine et ainsi induire une recombinaison de CSR optimale et fonctionnelle. Toutefois, nos données indiquent que ces core enhancers ne sont pas suffisants pour maintenir l’intégrité génomique de la cellule B comme en témoigne l’analyse des mutations sur des oncogènes tels que Bcl6. Ces travaux ouvrent de nouvelles perspectives pour la compréhension des mécanismes régissant le développement B tardif ainsi que le maintien de l’intégrité génomique via l’organisation du noyau.The late stages of B lymphocyte differentiation are characterized by gene rearrangements, such as somatic hypermutation and class recombination, which ultimately lead to the production of highly specific immunoglobulins. These processes, occurring at immunoglobulin-coding loci, introduce mutations and DNA double-strand breaks. Any malfunctioning of these rearrangements constitutes a major threat to the integrity of the B cell genome, and their regulation is therefore essential. Monitoring and control of these events are mediated by the cis-regulatory regions of the IgH locus: the EµMARs and 3'RR regions. The role of these regions at transcriptional level has been extensively characterized, using various mouse models with partial or total deletions of these regions. The spatio-temporal regulation of gene rearrangements has also been described at the nuclear level, with dynamic shifts of immunoglobulin loci, as well as at the supranucleosomal level through the formation of chromatin loops. Data from the laboratory and the literature suggest the involvement of cis-regulatory regions of the IgH locus in these additional regulatory mechanisms. We have demonstrated that core enhancers of the 3'RR are necessary and sufficient to correctly address the IgH locus in the nucleus and to allow chromatin loop formation and thus induce optimal and functional CSR recombination. However, our data indicate that these core enhancers are not sufficient to maintain the genomic integrity of the B cell, as shown by the analysis of mutations in oncogenes such as Bcl6. This work opens up new perspectives for understanding the mechanisms governing late B development and the maintenance of genomic integrity via nuclear organization

Contribution of Immunoglobulin Enhancers to B Cell Nuclear Organization

International audienceB 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

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

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Core enhancers of the 3’RR optimize IgH nuclear position and loop conformation for oriented CSR

Abstract Class switch recombination is an essential process which enabling B cells to adapt immunoglobulin subtypes to antigens. Transcription plays a crucial role in regulating CSR in which the IgH 3’Regulatory Region ( 3’RR ) was identified as a key player. The 3’RR stands at the 3’ end of IgH locus and is composed of four core enhancers surrounded by inverted repeated sequences, forming a quasi-palindrome. In addition to transcriptional control, nuclear organization appears to be an important level in CSR regulation. Furthermore, the chromatin loops at IgH locus facilitate an efficient CSR recombination by bringing the donor and acceptor switch regions closer together. However, the precise control mechanisms governing both of these processes remain partially understood. Here, using the reference DNA 3D-FISH technique combined with various high throughput approaches, we showed that 3’RR core enhancers are necessary and sufficient to preorganize resting B cell nuclei to facilitate a deletional CSR mechanism at activated stage. We demonstrated that the 3’RR core enhancers regulate IgH locus addressing in the nuclei, control IgH locus accessibility and orchestrate IgH loops formation. Our findings pinpointed an additional regulation level of mechanisms underlying B cell diversification. Graphical abstract Position of IgH loci through B cell development (from transitional to stimulated stages) is represented by the red spots. In wt and c3’RR context, IgH loci get closer from each other and from nucleus center during evolution from transitional to mature resting stage and they relocates more at nuclear periphery, away one from each other, upon in vitro stimulation. In Δ3’RR model, this dynamic is lost and, moreover, IgH loci are more localized to pericentromeric heterochromatin (represented by green area) since the mature resting B cell stage and remain in after in vitro stimulation

Core enhancers of the 3′RR optimize IgH nuclear position and loop conformation for successful oriented class switch recombination

International audienceAbstract In B lymphocytes, class switch recombination (CSR) is an essential process that adapts immunoglobulin (Ig) subtypes to antigen response. Taking place within the Ig heavy chain (IgH) locus, CSR needs controlled transcription of targeted regions governed by the IgH 3′ regulatory region (3′RR). This super-enhancer is composed of four core enhancers surrounded by inverted repeated sequences, forming a quasi-palindrome. In addition to transcription, nuclear organization appears to be an important level in CSR regulation. While it is now established that chromatin loop extrusion takes place within IgH locus to facilitate CSR by bringing the donor and acceptor switch regions closer together, the underlying mechanism that triggers CSR loop formation remains partially understood. Here, by combining DNA 3D fluorescence in situhybridization with various high-throughput approaches, we deciphered critical functions for the 3′RR core enhancer element in nuclear addressing, accessibility and chromatin looping of the IgH locus. We conclude that the 3′RR core enhancers are necessary and sufficient to pre-organize the position and conformation of IgH loci in resting B-cell nuclei to enable the deletional recombination events required for productive successful CSR in activated B-cell nuclei

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

Abstract SATB1 (Special A-T rich Binding protein 1) is a cell type specific factor involved in chromatin remodelling events that participate in the regulation of the genetic network in developing T cells and neurons. In T cells, SATB1 is a key factor required for lineage commitment, VDJ recombination, development and maturation. In B cells, SATB1 is described as binding to the MARs-Eµ regions of the IgH locus. Considering that its expression varies during differentiation, the involvement of this factor needed to be clarified in B cells. Using a KO mouse model deleting SATB1 from the pro-B cell stage, we were able to examine the consequences of SATB1 deletion in naive and activated B cell subsets. Our model indicates firstly that SATB1 is not essential for B cell development and the establishment of a broad IgH repertoire. Second, we show that this factor 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. This finding suggests that SATB1 may limit the introduction of unwanted mutations into B cells

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

International audienceIntroduction: Two scaffold/matrix attachment regions (5'-and 3'-MARs Eμ) flank the intronic core enhancer (cEμ) within the immunoglobulin heavy chain locus (IgH). Besides their conservation in mice and humans, the physiological role of MARs Eμ 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 MARs Eμ , further combined to relevant models deficient for base excision repair and mismatch repair. Results: We observed an inverted substitution pattern in of MARs Eμ-deficient animals: SHM being decreased upstream from cEμ and increased downstream of it. Strikingly, the SHM defect induced by MARs Eμ-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 MARs Eμ regions in limiting the error-prone repair machinery to the variable region of Ig gene loci