RESUMO
The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.
Assuntos
Doenças Cardiovasculares/metabolismo , Metilação de DNA , Miócitos Cardíacos/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/genética , Ilhas de CpG , DNA/química , Ecocardiografia , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos , Epigênese Genética , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Coração/embriologia , Histonas/química , Humanos , Camundongos , Fenótipo , Regiões Promotoras GenéticasRESUMO
During thymic T cell development, immature CD4(+)/CD8(+) thymocytes develop into either CD4(+)/CD8(-) helper or CD4(-)/CD8(+) CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8alpha strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4(-)/CD8(+) T cells. Instead, mature cells with a CD4(+)/CD8(+) phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation.
Assuntos
Antígenos CD4/genética , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Ligação a DNA/genética , Inativação Gênica/imunologia , Morfolinas/farmacologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Subpopulações de Linfócitos T/metabolismo , Timo/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Antígenos CD4/biossíntese , Antígenos CD4/metabolismo , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-7/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Subpopulações de Linfócitos T/citologia , Timo/citologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-gamma secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-gamma secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2.