RESUMEN
IgH class switch recombination (CSR) replaces Cµ constant region (CH) exons with one of six downstream CHs by joining transcription-targeted double-strand breaks (DSBs) in the Cµ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3'IgH regulatory region (3'IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are 10 consecutive CTCF-binding elements (CBEs) downstream of the 3'IgHRR, termed the "3'IgH CBEs." Prior studies showed that deletion of eight 3'IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3'IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except that of Sγ1. Moreover, deletion of all 3'IgH CBEs rendered the 6-kb region just downstream highly transcribed and caused sequences within to be aligned with Sµ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3'IgH CBEs as critical insulators for focusing loop extrusion-mediated 3'IgHRR transcriptional and CSR activities on upstream CH locus targets.
Asunto(s)
Factor de Unión a CCCTC/genética , Cambio de Clase de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Transcripción Genética/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Linfocitos B/inmunología , Cromatina/genética , Cromatina/inmunología , Mutación de Línea Germinal/genética , Cambio de Clase de Inmunoglobulina/inmunología , Ratones , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias Reguladoras de Ácidos Nucleicos/inmunologíaRESUMEN
RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6-9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteínas de Homeodominio/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Conformación de Ácido Nucleico , Animales , Linfocitos B/citología , Linfocitos B/enzimología , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Endonucleasas/deficiencia , Endonucleasas/genética , Puntos de Control de la Fase G1 del Ciclo Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Proteínas/genética , Proteínas/metabolismo , Recombinación V(D)J/genéticaRESUMEN
The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH-rearranged gene segment in the 3' proximal Igh domain. By using high-resolution mapping of long-range interactions, we demonstrate that local interaction domains established the three-dimensional structure of the extended Igh locus in lymphoid progenitors. In pro-B cells, these local domains engaged in long-range interactions across the Igh locus, which depend on the regulators Pax5, YY1, and CTCF. The large VH gene cluster underwent flexible long-range interactions with the more rigidly structured proximal domain, which probably ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. These long-range interactions appear to be an intrinsic feature of the VH gene cluster, because they are still generated upon mutation of the Eµ enhancer, IGCR1 insulator, or 3' regulatory region in the proximal Igh domain.
Asunto(s)
Diversidad de Anticuerpos/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Células Precursoras de Linfocitos B/inmunología , Animales , Secuencia de Bases , Sitios de Unión , Factor de Unión a CCCTC , Mapeo Cromosómico , Reordenamiento Génico , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX5/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , Factor de Transcripción YY1/metabolismoRESUMEN
CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Silenciador del Gen/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Proteínas Nucleares/antagonistas & inhibidores , Células Plasmáticas/citología , Proteínas Represoras/fisiología , Transactivadores/antagonistas & inhibidores , Animales , Subgrupos de Linfocitos B/citología , Células HEK293 , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Células Plasmáticas/inmunología , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Transactivadores/biosíntesis , Transactivadores/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Immunoglobulin heavy chain (IgH) variable region exons are assembled from V(H), D and J(H) gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the V(H) and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of D(H)-proximal V(H) gene segments and promoting rearrangement of distal V(H) segments. IGCR1 maintains ordered and lineage-specific V(H)(D)J(H) recombination by suppressing V(H) joining to D segments not joined to J(H) segments, and V(H) to DJ(H) joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal V(H)-to-DJ(H) recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.
Asunto(s)
ADN Intergénico/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Recombinación Genética/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/metabolismo , Exones VDJ/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Factor de Unión a CCCTC , Linaje de la Célula/genética , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Retroalimentación Fisiológica , Células Germinativas/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Mutación/genética , Timo/citología , Transcripción Genética/genéticaRESUMEN
Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5' to 3', several hundred V(H) gene segments, 13 D segments, and 4 J(H) segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J(H) rearrangement occurring on both alleles before appendage of a V(H) segment. Also, IgH V(H) to DJ(H) rearrangement does not occur in T cells, even though DJ(H) rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V(H) and D clusters (generating ΔV(H)-D alleles). In both B and T cells, ΔV(H)-D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V(H) segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J(H) rearrangement and substantial V(H) to DJ(H) rearrangement of proximal IgH V(H) segments. Thus, the V(H)-D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination.
Asunto(s)
Linfocitos B/metabolismo , Reordenamiento Génico de Cadena Pesada de Linfocito B/fisiología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , ARN sin Sentido/biosíntesis , Transcripción Genética/fisiología , Alelos , Animales , ADN Intergénico/genética , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Sitios Genéticos/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Mutantes , ARN sin Sentido/genética , Linfocitos T/metabolismoRESUMEN
Three antibacterial peptides, named protaetins 1, 2, and 3, were purified and characterized from immunized larval hemolymph of Protaetia brevitarsis, a fruit tree pest in Korea. Also, protaetin 1 was cloned. Acid extraction, gel filtration, preparative acid-urea PAGE, and reversed-phase FPLC were used for purification of peptides. Protaetins 1 and 3 had molecular masses of 7.5 and 12 kDa on Tricine SDS-PAGE, respectively, and the molecular mass of protaetin 2 was 9,283.95 Da as determined by MALDI-TOF mass spectrometry. In an antibacterial assay, protaetins showed antibacterial activities against a panel of Gram-positive and -negative bacteria. For the RT-PCR (reverse transcription polymerase chain reaction) to obtain the complete primary sequence, the primer was designed according to the N-terminal amino acid sequence of protaetin 1. Amino acid sequence homology of protaetin 1 with holotricin 2, an antibacterial peptide from Holotrichia diomphalia, showed 99% identity. Northern blot analysis showed that the protaetin 1 gene was strongly expressed in the fat body after Escherichia coli injection, but not in normal fat body. Also, it was expressed in the gut, but was much weaker after immunization.