Abstract
AbstractIt is not possible to clearly visualize how chromatin condenses to heterochromatin in vivo. However, in an in vitro system for Saccharomyces cerevisiae, the requirements for heterochromatin filament formation mirror those found in vivo. Here we report that the nucleosomes and the Sir2, Sir3 and Sir4 proteins, which are required for in vitro filament assembly, are also components of these filaments, confirming that the filaments are SIR-nucleosome filaments. We show the individual localization patterns of the Sir proteins on this SIR-nucleosome filament, and demonstrate that the metabolite, AAR, plays a specific and essential role in promoting the formation of this SIR-nucleosome filament.
Highlights
Sir2 and Sir4 are co-purified as a heterodimeric complex, which contains varying amounts of Sir3 [13, 19]
Sir2 couples this deacetylation to nicotinamide adenine dinucleotide (NAD) hydrolysis and generates the metabolite, O-acetyl-ADP-ribose (OAADPR, or AAR) [12, 17,18]. This deacetylation of histone amino terminal tails is followed by their association with Sir3 and Sir4, which leads to iterative recruitment cycles of silent information regulator (Sir) proteins along the adjacent chromatin fiber to form extended silent heterochromatin regions [13,14,15]
Both the deacetylation event and the AAR itself might contribute to SIR complex assembly in vitro, with both being required for the formation of silent heterochromatin in vivo
Summary
Sir2 and Sir4 are co-purified as a heterodimeric complex, which contains varying amounts of Sir3 [13, 19]. The Sir proteins form the SIR complex, capable of nucleosome binding [6,7,8,9], and which brings together two types of biochemical activities, critical for silent heterochromatin assembly. This deacetylation of histone amino terminal tails is followed by their association with Sir3 and Sir4, which leads to iterative recruitment cycles of Sir proteins along the adjacent chromatin fiber to form extended silent heterochromatin regions [13,14,15].
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