RESUMEN
Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.
Asunto(s)
Microdominios de Membrana/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Antígenos/metabolismo , Linfocitos B/enzimología , Proteínas de Unión al ADN , Humanos , Inmunoglobulina M/genética , Inmunoglobulina M/metabolismo , Lipoilación , Activación de Linfocitos , Microdominios de Membrana/enzimología , Ratones , Mutación/genética , Oncogenes , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción TFII/metabolismo , Transcripción GenéticaRESUMEN
Bright/Dril1/ARID3a is a B cell-specific, matrix association (or attachment) region-binding transcriptional regulator of immunoglobulin heavy chain genes and of E2F1-dependent cell cycle progression. Bright contains a central DNA binding domain termed ARID (AT-rich interacting domain) and a C-terminal region termed REKLES (for a conserved amino acid motif). The ARID domain has been identified in seven highly conserved families of metazoan proteins (ARID1-5 and JARID1-2), whereas REKLES is found only in the ARID3 subfamily (composed of Bright/ARID3a, Bdp/ARID3b, and Bright-like/ARID3c). REKLES consists of two subdomains: a modestly conserved N-terminal REKLESalpha and a highly conserved (among ARID3 orthologous proteins) C-terminal REKLESbeta. Previously we showed that Bright undergoes nucleocytoplasmic shuttling and that REKLESalpha and -beta were required, respectively, for nuclear import and Crm1-dependent nuclear export. Here we show that Bright further requires REKLESbeta for self-association or paralogue association and for nuclear matrix targeting. REK-LES promotes and regulates the extent of Bright multimerization, which occurs in the absence or presence of target DNA and is necessary for specific DNA binding. REKLESbeta-mediated interaction of Bright with Bdp, which localizes strictly to the nucleus, traps Bright within the nucleus via neutralization of its nuclear export activity. These results identify REKLES as a multifunctional domain that has co-evolved with and regulates functional properties of the ARID3 DNA binding domain.
Asunto(s)
Proteínas de Unión al ADN/genética , Evolución Molecular , Oncogenes/genética , Transactivadores/genética , Transporte Activo de Núcleo Celular/fisiología , Secuencias de Aminoácidos/genética , Animales , Linfocitos B/metabolismo , Células COS , Chlorocebus aethiops , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Matriz Nuclear/genética , Matriz Nuclear/metabolismo , Especificidad de Órganos/fisiología , Estructura Terciaria de Proteína/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Factores de TranscripciónRESUMEN
BACKGROUND: Chromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development. RESULTS: Alternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles. CONCLUSION: We propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.
Asunto(s)
Proteínas Portadoras/metabolismo , Centro Germinal/metabolismo , Linfoma de Células B/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Empalme Alternativo/genética , Animales , Western Blotting , Células COS , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Perfilación de la Expresión Génica , Centro Germinal/patología , Células HeLa , Humanos , Inmunoprecipitación , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/análisis , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas RepresorasRESUMEN
The ARID is an ancient DNA-binding domain that is conserved throughout the evolution of higher eukaryotes. The ARID consensus sequence spans about 100 amino acid residues, and structural studies identify the major groove contact site as a modified helix-turn-helix motif. ARID-containing proteins exhibit a range of cellular functions, including participation in chromatin remodeling, and regulation of gene expression during cell growth, differentiation, and development. A subset of ARID family proteins binds DNA specifically at AT-rich sites; the remainder bind DNA nonspecifically. Orthologs to each of the seven distinct subfamilies of mammalian ARID-containing proteins are found in insect genomes, indicating the minimum age for the organization of these higher metazoan subfamilies. Many of these ancestral genes were duplicated and fixed over time to yield the 15 ARID-containing genes that are found in the human, mouse, and dog genomes. This paper describes a nomenclature, recommended by the Mouse Genomic Nomenclature Committee (MGNC) and accepted by the Human Genome Organization (HUGO) Gene Nomenclature Committee, for these mammalian ARID-containing genes that reflects this evolutionary history.
