RESUMO
A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-beta locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-beta gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-beta chromatin was resistant to DNase I digestion in these cells. Thus, the collaboration is shown to convert the Ig-beta chromatin from the condensed state to a relaxed state. H3 and H4 acetylation decreased to <8% but H3K4 hypermethylation was observed at the Ig-beta promoter and exon 3. The collaboration of four regions had virtually no effect on CG hypomethylation in the region upstream the transcriptional start site. Accordingly, neither the DNase I general sensitive state in the Ig-beta chromatin nor hyperacetylation of H3 and H4 histones in the promoter proximal region causes H3K4 di-methylation or CG hypomethylation in the promoter. From these analyses, a chromatin situation was found in which both an active state, such as enhanced H3K4 methylation, or CG hypomethylation, and an inactive state, such as DNase I resistance in the Ig-beta chromatin or hypoacetylation of H3 and H4 histones in the Ig-beta locus, coexist.
Assuntos
Antígenos CD79/genética , Galinhas/genética , Cromatina/química , Elementos Reguladores de Transcrição , Ativação Transcricional , Acetilação , Animais , Antígenos CD79/metabolismo , Linhagem Celular , Desoxirribonuclease I , Éxons , Histonas/metabolismo , Metilação , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Deleção de Sequência , Transcrição GênicaRESUMO
The role of DNase I hypersensitive sites (DHSs) in transcription of the B cell-specific Ig-beta gene and in maintenance of active chromatin state in the Ig-beta locus were examined. A total of 10 DHSs were divided into four regions, and each region was deleted separately in chicken B lymphocyte-derived DT40 cells. Deletion of three DHSs located between the Ig-beta promoter and its upstream Na channelgene, resulted in the absence of Ig-beta mRNA. Three regions except the region in the Na channel gene were involved in the transcription of Ig-beta gene. The enhancing activity of DHSs as determined by transient transfection assays did not always correlate with the effect of DHS deletion on the expression level of Ig-beta mRNA. In each deletion, cells contained the same DHSs as observed in the predeletion cells, indicating that deleted DHSs did not participate in the maintenance of DT40-specific DHSs. Enhanced acetylation of H3 and H4 histones at the Ig-beta promoter and at DT40-specific DHSs was observed in cells in which DHSs between the Na channel gene and Ig-beta promoter were deleted; therefore, these DHSs are prerequisite for transcription of the Ig-beta gene but not required for the maintenance of active chromatin state in the Ig-beta locus. Thus, epigenetic factors required for the maintenance of the active chromatin state are suggested to reside in other regions than those deleted in this study.
Assuntos
Antígenos CD/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Desoxirribonuclease I/metabolismo , Transcrição Gênica , Acetilação , Animais , Antígenos CD79 , Galinhas , Hormônio do Crescimento/genética , Histonas/metabolismoRESUMO
DNase I hypersensitive sites (DHSs) and histone acetylation status were examined in the Ig-beta locus of chicken B lymphocyte-derived DT40 cells and liver-derived LMH cells. Twelve DT40-specific DHSs were identified: one in the Ig-beta promoter, one in the first intron of the Ig-beta gene, three in the sodium channel gene located upstream of the Ig-beta gene, two between the sodium channel gene and the Ig-beta gene, four between the Ig-beta gene and a downstream growth hormone (GH) gene, and one in the downstream region of the GH gene. Transient transfection studies show that the DHS in the intron of Ig-beta gene enhances the activity of the Ig-beta promoter fourfold. A 1.6 kb DNA fragment, which includes two DHSs, from the sodium channel gene enhanced promoter activity threefold. The transcription enhancing ability of the intron DHS was dependent on orientation, but was not promoter specific. Electrophoretic mobility shift assays (EMSA) demonstrated that an Ets protein family member binds to the intron DHS. In DT40 cells, a distinguished acetylation of H3 and H4 histones was found at the Ig-beta promoter, in addition to the enhanced acetylation of both histones at DT40-specific DHSs.
Assuntos
Antígenos CD/genética , Desoxirribonuclease I/metabolismo , Histonas/metabolismo , Acetilação , Animais , Antígenos CD/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Antígenos CD79 , Linhagem Celular , Galinhas , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Hormônio do Crescimento/genética , Luciferases/genética , Luciferases/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canais de Sódio/genética , Transcrição Gênica/genética , TransfecçãoRESUMO
Using Ig-beta and growth hormone producing cells with liver-derived cells for controls, sensitivity of chromatin to DNase I was measured by real-time PCR at eleven targets in rat Ig-beta/growth hormone locus where four cell type-specific genes and two ubiquitously expressed genes are present in a compact 88-kb region. Chromatin situated at the promoter of actively-transcribed gene and placed at cell type-specific DNase I hypersensitive sites with enhancer activity was sensitive to DNase I. In the case of inactive gene, chromatin located in these regions was resistant to DNase I. Unexpectedly, however, chromatin placed in the transcribed intron was resistant to DNase I in two genes. DNase I sensitive chromatin was shown not to distribute locus-widely but rather to localize at the promoter and the enhancer of actively-transcribed genes in this locus.
Assuntos
Cromatina/metabolismo , Desoxirribonuclease I/metabolismo , Hormônio do Crescimento/metabolismo , Imunoglobulinas/metabolismo , Animais , Células Cultivadas , Cromatina/genética , Elementos Facilitadores Genéticos , Éxons , Hormônio do Crescimento/genética , Imunoglobulinas/genética , Íntrons , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RatosRESUMO
The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) at 11 targets in the rat Ig-beta/growth hormone locus. Marked enhancement of the acetylation of histones H3 and H4 and the dimethylation of H3 Lys4 was observed in the chromatin situated close to the promoter of an actively transcribed gene. Chromatin positioned near a cell-type-specific DNase I-hypersensitive site with enhancer activity had the same histone modifications as the active promoter. In one transcribed intron, chromatin with fewer histone modifications was found, and in another transcribed intron, chromatin with markedly enhanced modifications was found. In most cases, no appreciable difference in the acetylation of histones H3 and H4 was found at prominently enhanced targets. However, different acetylation levels of H3 and H4 were found at one target. The targets with enhanced dimethylation of the H3 Lys4 residue coincided with those with prominently enhanced acetylation of histones H3 and H4.
