RESUMO
BCL6 encodes a transcriptional repressor that is essential for the germinal center (GC) reaction and important in lymphomagenesis. Although its promoter has been well studied, little is known concerning its possible regulation by more distal elements. To gain such information, we mapped critical histone modifications associated with active transcription within BCL6 as well as far upstream sequences at nucleosomal resolution in B-cell lines and in normal naive and GC B cells. Promoter-associated and intronic CpG islands (CGIs) in BCL6 showed a reciprocal pattern of histone modifications. Gene expression correlated with a paradoxical loss from the intronic CGI of histone H3 lysine-4 trimethylation, normally associated with transcription, suggesting that the intronic CGI may interfere with transcription. In an approximately 110-kb region extending 150-260 kb upstream of BCL6, highly active histone modifications were present only in normal GC B cells and a GC B-cell line; this region overlaps with an alternative breakpoint region for chromosomal translocations and contains a GC-specific noncoding RNA gene. By chromosome conformation capture, we determined that the BCL6 promoter interacts with this distant upstream region. It is likely that transcriptional enhancers in this region activate BCL6 and overcome strong autorepression in GC B cells.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/genética , Centro Germinativo/metabolismo , Histonas/química , Regiões Promotoras Genéticas , Linfócitos B/citologia , Linfócitos B/imunologia , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Sequência Conservada , Proteínas do Citoesqueleto/genética , Metilação de DNA , Primers do DNA/genética , Proteínas de Ligação a DNA/imunologia , Epigênese Genética , Expressão Gênica , Centro Germinativo/imunologia , Histonas/metabolismo , Humanos , Íntrons , Proteínas com Domínio LIM , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Repressoras/genética , Proteínas Repressoras/imunologiaRESUMO
A cluster of six microRNAs (miRNAs), miR-17-92, is processed from the transcript of C13orf25, a gene amplified in some lymphomas and solid tumors. We find that levels of the miRNAs in the cluster do not vary entirely in parallel with each other or with the primary RNA in B-cell lines or normal cells, suggesting that processing or stability of the miRNAs is differentially regulated. Using luciferase reporter assays, we identified the region required for maximum promoter activity. Additional deletions and mutations indicated that the promoter is regulated by the collaborative activity of several transcription factors, most of which individually have only a moderate effect; mutation of a cluster of putative SP1-binding sites, however, reduces promoter activity by 70%. MYC is known to regulate C13orf25; surprisingly, mutation of a putative promoter MYC-binding site enhanced promoter activity. We found that the inhibitory MYC family member MXI1 bound to this region. The chromatin structure of a >22.5-kb region encompassing the gene contains peaks of activating histone marks, suggesting the presence of enhancers, and we confirmed that at least two regions have enhancer activity. Because the miR-17-92 cluster acts as an important oncogene in several cancers and targets genes important in regulating cell proliferation and survival, further studies of its transcriptional control are warranted.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma/genética , MicroRNAs/genética , Família Multigênica/genética , Animais , Linfócitos B , Pareamento de Bases/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Sequência Conservada/genética , Elementos E-Box/genética , Loci Gênicos/genética , Células HEK293 , Histonas/metabolismo , Humanos , Luciferases de Vaga-Lume/metabolismo , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Longo não Codificante , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Proteínas Supressoras de Tumor/metabolismoRESUMO
BCL6 gene transcribes two main mRNA isoforms, variant 1 and variant 2, which have distinct transcription start sites. However, these two isoforms encode identical BCL6 protein. Recently, a third variant was sequenced from a human hippocampus cDNA library (DB465062). In this study, we cloned and sequenced a novel BCL6 spliced isoform (BCL6S) which lacks exon 7 that encodes the first two zinc fingers of BCL6. We found a splicing isoform, BCL6S, that excludes exon 7 but retains the last four of the six zinc fingers (ZFs) coding region of the BCL6 gene. BCL6S mRNA and protein were expressed in human cell lines and tissues that expressed BCL6, but accounted for a minor portion of BCL6 transcripts or protein. BCL6S protein was also detectable in BCL6 positive cells. BCL6S could form homodimers or heterodimers with BCL6 and could bind to classical BCL6-binding sites. Luciferase reporter assays demonstrated that BCL6S could effectively repress typical BCL6 target genes. BCL6S is a compact repressor that may have a functional role in normal and neoplastic germinal center B cells.