CCCTC-binding factor locks premature IgH germline transcription and restrains class switch recombination

In response to antigenic stimulation B cells undergo class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) to replace the primary IgM/IgD isotypes by IgG, IgE, or IgA. CSR is initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues at the switch (S) regions of IgH. B cell stimulation promotes germline transcription (GLT) of specific S regions, a necessary event prior to CSR because it facilitates AID access to S regions. Here, we show that CCCTC-binding factor (CTCF)-deficient mice are severely impaired in the generation of germinal center B cells and plasma cells after immunization in vivo, most likely due to impaired cell survival. Importantly, we find that CTCF-deficient B cells have an increased rate of CSR under various stimulation conditions in vitro. This effect is not secondary to altered cell proliferation or AID expression in CTCF-deficient cells. Instead, we find that CTCF-deficient B cells harbor an increased mutation frequency at switch regions, probably reflecting an increased accessibility of AID to IgH in the absence of CTCF. Moreover, CTCF deficiency triggers premature GLT of S regions in naïve B cells. Our results indicate that CTCF restricts CSR by enforcing GLT silencing and limiting AID access to IgH

Estrés en el personal de enfermería

El siguiente trabajo de investigación tiene como objetivo principal indagar, conocer e identificar cuáles son los factores estresores que más afectan al personal de enfermería de servicios abiertos y cerrados (Clínica Medica y Unidad de Terapia Intensiva) del Hospital Alfredo I. Perrupato del departamento de San Martin, Mendoza, en el primer semestre del 2015. Es un estudio de tipo cuantitativo, descriptivo y de corte transversal. Se tomó una muestra de 49 enfermeros que trabajan en dichos servicios a los que se les realizó una encuesta anónima con preguntas cerradas de múltiple opción. Las variables que se evaluaron fueron características del personal (sexo, edad, estado civil, etc.), laborales (servicio, anos de trabajo, turnos, horas de trabajo, etc.) y desgaste físico (presencia de cefaleas, fatiga, temor, etc.)Fil: Montivero, Marina Soledad. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Escuela de Enfermería..Fil: Reinoso, Delia Ester. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Escuela de Enfermería..Fil: Zárate, Raquel Marisol. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Escuela de Enfermería.

CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation

Activation-induced deaminase expression defines mature B cell lymphoma in the mouse

Germinal centers (GCs) are the sites of secondary antibody diversification and underlie the mechanism of action of many vaccination strategies. Activation-induced deaminase (AID) triggers secondary antibody diversification through the introduction of somatic changes in immunoglobulin genes which lead to the generation of antibodies of higher affinity and more specialized effector functions. However, AID can also target other genomic regions, giving rise to mutations and chromosome translocations with oncogenic potential. Many human lymphomas originate from mature B cells that have undergone the GC reaction, such as the diffuse large B cell lymphoma, the follicular lymphoma and Burkitt lymphoma, and carry chromosome translocations. Mature B cell lymphomagenesis has been modeled in the mouse by the genetic introduction of chromosome translocations. Here, we present an in-depth characterization of one such model, λ-MYC mice. We found that young pre-tumor stage mice had a prominent block in early B cell differentiation that resulted in the generation of very aggressive tumors lacking surface B cell receptor (BCR) expression, indicating that a large fraction of tumors in λ-MYC mice arise from B cell precursors rather than from mature B cells. Further, we assessed the contribution of AID to B cell lymphomagenesis in λ-MYC mice by using a genetic tracer of historical AID expression. Only a fraction of tumors contained cells of GC origin as defined by AID expression. AID-experienced tumors associated with longer survival and resembled mature B cell lymphomas. Thus, AID expression defines Burkitt lymphomagenesis in λ-MYC mice

CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation

CTCF regulates transcriptional programs and antibody diversification in mature B cells

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-09-2020Esta tesis tiene embargado el acceso al texto completo hasta el 25-03-2022Durante la respuesta inmune los linfocitos B maduros diversifican sus genes de inmunoglobulinas (Igs) en los centros germinales (GCs), dando lugar a células plasmáticas de alta afinidad y células B de memoria. Esta diversificación secundaria viene de las reacciones hipermutación somática (SHM) y del de cambio de isotipo (CSR), que se inician por la Desaminasa Inducida por Activación (AID). La reacción de centro germinal conlleva una compleja regulación transcripcional que permite coordinar la expresión de genes implicados en proliferación y supervivencia celular, daño en el ADN, señalización o diferenciación terminal. CTCF es un remodelador de cromatina implicado en la generación de lazos en el ADN que permiten establecer interacciones entre diferentes dominios en la cromatina, actuando como regulador de la expresión génica. En células B, CTCF es fundamental durante la recombinación V(D)J en la diferenciación temprana de linfocitos B en la médula ósea, al favorecer interacciones de regiones distantes del ADN. Al igual que en la recombinación V(D)J, el CSR está estrechamente ligado a la transcripción e implica la formación de interacciones entre elementos reguladores alejados en el ADN. Esta tesis doctoral se ha centrado en establecer el papel de CTCF en la reacción de centro germinal así como durante la diversificación secundaria de anticuerpos. Para ello se generó un modelo condicional murino en el que se elimina de forma específica CTCF en células B maduras (CTCFfl/fl CD19-Creki/+). Encontramos que CTCF es esencial para la reacción de GC in vivo y que es un regulador maestro del programa transcripcional que se desencadena durante la activación de las células B. Sorprendentemente, observamos que la deficiencia de CTCF aumenta la eficiencia en el CSR sin alterar la capacidad proliferativa de las células B ni la expresión de AID. Sin embargo, las células deficientes en CTCF tienen mayor frecuencia de SHM y más transcritos germinales en las regiones de switch de la cadena pesada de inmunoglobulinas (IgH), lo que sugiere que en ausencia de CTCF está aumentada la accesibilidad de AID a esta región. De acuerdo con estas observaciones, encontramos una disminución de las interacciones entre los elementos reguladores del locus de IgH, lo que indica que la conformación tridimensional del locus IgH está alterada en ausencia de CTCF. Nuestros datos sugieren que CTCF es un regulador negativo del CSR, ya que controla el cambio arquitectónico del locus de inmunoglobulinas, presumiblemente evitando el acceso y mutagénesis prematura de AID. Por lo tanto, en este trabajo hemos establecido que CTCF es un regulador esencial de los cambios transcripcionales necesarios para la diferenciación terminal de las células B y de la diversificación secundaria de anticuerposDuring the immune response B cells engage in the germinal center (GC) reaction and undergo secondary antibody diversification by somatic remodeling of their immunoglobulin (Ig) genes, resulting in the generation of high affinity memory B cells or antibody-secreting plasma cells. These diversification events are called somatic hypermutation (SHM) and class switch recombination (CSR) and are both initiated by activation-induced cytidine deaminase (AID). The biology of GCs is extremely complex and entails proliferation, survival, cell death, DNA damage response and cell fate decisions. CTCF is a key chromatin remodeler involved in establishing long-range interactions that define chromatin architecture and functional domains, which, in turn, regulate the cell transcriptional program. CTCF is critical during V(D)J recombination early in B cell differentiation in the bone marrow by favoring interactions of distal DNA regions. Like V(D)J recombination, CSR is linked to transcription and involves the formation of long-range DNA loops that facilitate the interaction between distant cis regulatory regions. Here, we have assessed the regulatory role of CTCF during the GC and during antibody diversification. We made use of a conditional mouse where CTCF is eliminated specifically in mature B cells (CTCFfl/fl CD19-Creki/+). We found that CTCF is essential for the GC response in vivo and is a key regulator of the transcriptional program triggered by B cell activation. Unexpectedly, we observed that CTCF deficient B cells switch more efficiently than control cells without any concomitant alterations in cell proliferation or AID expression. However, in CTCF deficient B cells, both SHM frequency and germline transcription are increased in the IgH switch regions, suggesting that the absence of CTCF facilitates the access of AID to this region. In agreement with these observations, we found decreased interactions between regulatory elements at the IgH locus, indicating that the three-dimensional conformation of the IgH locus is altered in absence of CTCF. Our data suggest that CTCF is a negative regulator of the CSR, possibly by controlling the architectural changes at IgH and preventing AID from accessing and mutating prematurely the switch regions. Therefore, in this work we have established that CTCF plays essential roles in the transcriptional changes involved in the terminal differentiation of B cells and in the secondary diversification of antibodie

Low-affinity CTCF binding drives transcriptional regulation whereas high-affinity binding encompasses architectural functions.

CTCF is a DNA-binding protein which plays critical roles in chromatin structure organization and transcriptional regulation; however, little is known about the functional determinants of different CTCF-binding sites (CBS). Using a conditional mouse model, we have identified one set of CBSs that are lost upon CTCF depletion (lost CBSs) and another set that persists (retained CBSs). Retained CBSs are more similar to the consensus CTCF-binding sequence and usually span tandem CTCF peaks. Lost CBSs are enriched at enhancers and promoters and associate with active chromatin marks and higher transcriptional activity. In contrast, retained CBSs are enriched at TAD and loop boundaries. Integration of ChIP-seq and RNA-seq data has revealed that retained CBSs are located at the boundaries between distinct chromatin states, acting as chromatin barriers. Our results provide evidence that transient, lost CBSs are involved in transcriptional regulation, whereas retained CBSs are critical for establishing higher-order chromatin architecture.We thank all the members of the B lymphocyte Biology lab for helpful suggestions, Sonia Mur for technical assistance, Ana Losada and Ana Cuadrado for helpful discussions on experimental design, Roma´ n Pe´ rez Santalla for his help on algorithm design, Simon Bartlett for English editing, and the CNIC Genomics Unit for ChIP-seq and RNA-seq. We also thank Rafael Casellas and Erez Aiden for sharing HiC data with us and Jing Luan and Gerd Blobel for their help to access their datasets. This work was supported by grants from the Spanish Ministerio de Economı´a, Industria y Competitividad and ERDF, A way of making Europe (SAF2016-75511-R), the Spanish Ministerio de Ciencia e Innovacio´ n (PID2019-106773RB-I00/AEI/10.13039/ 501100011033) and the ‘‘la Caixa’’ Banking Foundation under the project code HR17-00247 to A.R.R. F.S.-C. received support from the Spanish Ministerio de Economı´a y Competitividad (RTI2018-102084-B-I00). E.M.-Z. and A.R.-R. are fellows of the research training program (FPI) funded by the Ministerio de Economı´a y Competitividad (BES-2014-069525) and Ministerio de Ciencia e Innovacio´ n (PRE2020-091873). M.J.G., F.S.-C., and A.R.R. are supported by CNIC. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacio´ n (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence, CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033.S

Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates

Abstract Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation