RESUMO
The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.
Assuntos
Encéfalo/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Metaboloma/fisiologia , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Comportamento Alimentar , Feminino , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/análise , Longevidade , Masculino , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismoRESUMO
Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.