Assuntos
Anticorpos Monoclonais/farmacologia , Dermatite/tratamento farmacológico , Receptores de Interleucina-17/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Ensaios Clínicos como Assunto , Humanos , Injeções SubcutâneasRESUMO
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
Using avian B lymphoma-derived DT40 cells, we disrupted a gene encoding the transcription factor PU.1. The mutant mRNA codes for a protein incapable of functioning as a transcription factor because of the deletion of the protein's DNA-binding domain. The absence of a functional PU.1 protein in the mutant cells was confirmed by Western blotting and electrophoretic mobility shift assay, thereby demonstrating that PU.1 was not essential for the proliferation of DT40 cells. An examination of the expression of several genes known to be PU.1 protein targets revealed almost the same levels of Ig-beta and Ig lambda L chain mRNA in mutant cells as in wild-type cells, indicating that the PU.1 protein plays no essential role in the transcription of these genes. Mutant cell doubling times 1.3 times longer than those of wild-type cells confirmed the PU.1 protein to be involved in the proliferation of B lymphocytes.
