RESUMO
Human T(H)2 cell differentiation results in the selective demethylation of several specific CpG dinucleotides in the IL-4 and IL-13 genes, which are expressed in activated T(H)2, but not T(H)1, cells. This demethylation is accompanied by the appearance of six DNase I hypersensitive sites within 1.4 kb at the 5'-end of the IL-4 gene. Micrococcal nuclease (MNase) digestion revealed that in both T(H)1 and T(H)2 cells nine nucleosomes with a repeat length of 201 bp are identically positioned around the 5'-end of the IL-4 gene. However, only in T(H)2 cells are six out of the eight intervening linkers exposed to DNase I. This suggests that a major perturbation of the higher-order chromatin structure occurs above the level of the nucleosome in vivo. It is observed in cells that are poised for expression but which are not actively expressing the gene (i.e. resting T(H)2 cells). Notably, all the demethylated CpGs in T(H)2 cells are found in DNA that is accessible to DNase I. This may suggest that the opening of the chromatin structure allows binding of specific trans-acting factors that prevent de novo methylation.
Assuntos
Cromatina/química , Metilação de DNA , Interleucina-4/genética , Células Th2/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 5 , Ilhas de CpG/fisiologia , DNA/química , Desoxirribonuclease I/metabolismo , Humanos , Células Jurkat , Nuclease do Micrococo/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Nucleossomos/química , Análise de Sequência de DNA , Sulfitos/farmacologiaRESUMO
The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.
Assuntos
Caenorhabditis elegans/fisiologia , Dano ao DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans , DNA/efeitos da radiação , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Embrião não Mamífero , Deleção de Genes , Genes Letais , Células Germinativas/química , Células Germinativas/metabolismo , Células Germinativas/efeitos da radiação , Dados de Sequência Molecular , Rad51 Recombinase , Proteína de Replicação ARESUMO
The differentiation of naive CD4(+) T lymphocytes into Th1 and Th2 lineages generates either cellular or humoral immune responses. Th2 cells express the cytokines IL-4, -5, and -13, which are implicated in asthma and atopy. Much has been published about the regulation of murine Th2 cytokine expression, but studies in human primary T cells are less common. We have developed a method for differentiating human CD45RA(+) (naive) T cells into Th1 and Th2 populations that display distinct cytokine expression profiles. We examined both CpG methylation, using bisulfite DNA modification and sequencing, and chromatin structure around the IL-4 and IL-13 genes before and after human T cell differentiation and in normal human skin fibroblasts. In naive cells, the DNA was predominantly methylated. After Th2 differentiation, DNase I hypersensitive sites (DHS) appeared at IL-4 and IL-13 and CpG demethylation occurred only around the Th2-specific DHS. Both DHS and CpG demethylation coincided with consensus binding sites for the Th2-specific transcription factor GATA-3. Although fibroblasts, like naive and Th1 cells, did not express IL-4 or IL-13, DHS and unmethylated CpG sites that were distinct from the Th2-specific sites were observed, suggesting that chromatin structure in this cluster not only varies in T cells according to IL-4/IL-13 expression but is also tissue specific.