ABSTRACT
The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.
Subject(s)
Chromatin Assembly and Disassembly , Cryoelectron Microscopy , DNA-Binding Proteins , Nucleosomes , Cryoelectron Microscopy/methods , Nucleosomes/chemistry , Nucleosomes/metabolism , Nucleosomes/ultrastructure , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Models, Molecular , Protein Binding , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , DNA/chemistry , DNA/metabolism , Histones/chemistry , Histones/metabolism , Histones/genetics , Humans , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription Factors/genetics , Adenosine Triphosphatases , Chromosomal Proteins, Non-Histone , Trans-ActivatorsABSTRACT
In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP. These structures suggest that Rpd3S utilizes a conserved Sin3 basic surface to navigate through the nucleosomal DNA, guided by its interactions with H3K36 methylation and the extra-nucleosomal DNA linkers, to target acetylated H3K9 and sample other histone tails. Furthermore, our structures illustrate that Rpd3S reconfigures the DNA linkers and acts in concert with Hho1 to engage the NCP, potentially unraveling how Rpd3S and Hho1 work in tandem for gene silencing.
