RESUMO
All eukaryotic cells divide a finite number of times, although the mechanistic basis of this replicative aging remains unclear. Replicative aging is accompanied by a reduction in histone protein levels, and this is a cause of aging in budding yeast. Here we show that nucleosome occupancy decreased by 50% across the whole genome during replicative aging using spike-in controlled micrococcal nuclease digestion followed by sequencing. Furthermore, nucleosomes became less well positioned or moved to sequences predicted to better accommodate histone octamers. The loss of histones during aging led to transcriptional induction of all yeast genes. Genes that are normally repressed by promoter nucleosomes were most induced, accompanied by preferential nucleosome loss from their promoters. We also found elevated levels of DNA strand breaks, mitochondrial DNA transfer to the nuclear genome, large-scale chromosomal alterations, translocations, and retrotransposition during aging.
Assuntos
Envelhecimento/genética , Genoma Fúngico/genética , Instabilidade Genômica/genética , Nucleossomos/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação para Cima , Aberrações Cromossômicas , Quebras de DNA , DNA Mitocondrial/genética , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Regiões Promotoras Genéticas/genética , TATA Box/genéticaRESUMO
The causal relationship between genomic instability and replicative aging is unclear. We reveal here that genomic instability at the budding yeast ribosomal DNA (rDNA) locus increases during aging, potentially due to the reduced cohesion that we uncovered during aging caused by the reduced abundance of multiple cohesin subunits, promoting increased global chromosomal instability. In agreement, cohesion is lost during aging at other chromosomal locations in addition to the rDNA, including centromeres. The genomic instability in old cells is exacerbated by a defect in DNA double-strand break (DSB) repair that we uncovered in old yeast. This was due to limiting levels of key homologous recombination proteins because overexpression of Rad51 or Mre11 reduced the accumulation of DSBs and largely restored DSB repair in old cells. We propose that increased rDNA instability and the reduced DSB repair capacity of old cells contribute to the progressive accumulation of global chromosomal DNA breaks, where exceeding a threshold of genomic DNA damage ends the replicative life span.
Assuntos
Replicação do DNA/genética , Recombinação Homóloga/genética , Saccharomycetales/genética , Saccharomycetales/fisiologia , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Ribossômico/genética , Loci Gênicos , Genoma Fúngico , Instabilidade Genômica , RNA não Traduzido/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/citologia , Transcrição GênicaRESUMO
DNA replication is a highly conserved process that accurately copies the genetic information from one generation to the next. The processes of chromatin disassembly and reassembly during DNA replication also have to be precisely regulated to ensure that the genetic material is compactly packaged to fit into the nucleus while also maintaining the epigenetic information that is carried by the histone proteins bound to the DNA, through cell divisions. Half of the histones that are deposited during replication are from the parental chromatin and carry the parental epigenetic information, while the other half of the histones are newly-synthesized. It has been of growing interest to understand how the parental pattern of epigenetic marks is re-established on the newly-synthesized histones, in a DNA sequence-specific manner, in order to maintain the epigenetic information through cell divisions. In this review we will discuss how histone chaperone proteins precisely coordinate the chromatin assembly process during DNA replication. We also discuss the recent evidence that histone-modifying enzymes, rather than the parental histones, are themselves epigenetic factors that remain associated with the DNA through replication to re-establish the epigenetic information on the newly-assembled chromatin.
RESUMO
BACKGROUND: The protein anti-silencing function 1 (Asf1) chaperones histones H3/H4 for assembly into nucleosomes every cell cycle as well as during DNA transcription and repair. Asf1 interacts directly with H4 through the C-terminal tail of H4, which itself interacts with the docking domain of H2A in the nucleosome. The structure of this region of the H4 C-terminus differs greatly in these two contexts. RESULTS: To investigate the functional consequence of this structural change in histone H4, we restricted the available conformations of the H4 C-terminus and analyzed its effect in vitro and in vivo in Saccharomyces cerevisiae. One such mutation, H4 G94P, had modest effects on the interaction between H4 and Asf1. However, in yeast, flexibility of the C-terminal tail of H4 has essential functions that extend beyond chromatin assembly and disassembly. The H4 G94P mutation resulted in severely sick yeast, although nucleosomes still formed in vivo albeit yielding diffuse micrococcal nuclease ladders. In vitro, H4G4P had modest effects on nucleosome stability, dramatically reduced histone octamer stability, and altered nucleosome sliding ability. CONCLUSIONS: The functional consequences of altering the conformational flexibility in the C-terminal tail of H4 are severe. Interestingly, despite the detrimental effects of the histone H4 G94P mutant on viability, nucleosome formation was not markedly affected in vivo. However, histone octamer stability and nucleosome stability as well as nucleosome sliding ability were altered in vitro. These studies highlight an important role for correct interactions of the histone H4 C-terminal tail within the histone octamer and suggest that maintenance of a stable histone octamer in vivo is an essential feature of chromatin dynamics.