Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF.
Chromatin remodeling enzymes use the energy of ATP hydrolysis to alter histone-DNA contacts and regulate DNA-based processes in eukaryotes. Whether different subfamilies of remodeling complexes generate distinct products remains uncertain. We have developed a protocol to analyze nucleosome remodeling on individual products formed in vitro. We used a DNA methyltransferase to examine DNA accessibility throughout nucleosomes that had been remodeled by the ISWI and SWI/SNF families of enzymes. We confirmed that ISWI-family enzymes mainly created patterns of accessibility consistent with canonical nucleosomes. In contrast, SWI/SNF-family enzymes generated widespread DNA accessibility. The protection patterns created by these enzymes were usually located at the extreme ends of the DNA and showed no evidence for stable loop formation on individual molecules. Instead, SWI/SNF family proteins created extensive accessibility by generating heterogeneous products that had fewer histone-DNA contacts than a canonical nucleosome, consistent with models in which a canonical histone octamer has been 'pushed' off of the end of the DNA.
Bouazoune
Karim
K
Department of Molecular Biology & Genetics, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Miranda
Tina B
TB
Jones
Peter A
PA
Kingston
Robert E
RE
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000378:metabolism; D015257:DNA-Cytosine Methylases; D006657:Histones / Q000737:chemistry; D009687:Nuclear Proteins / Q000378:metabolism; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D002868:Chromosomal Proteins, Non-Histone; D004247:DNA; D004265:DNA Helicases; D015257:DNA-Cytosine Methylases; D006657:Histones; D009687:Nuclear Proteins; D009707:Nucleosomes; D014157:Transcription Factors; C492319:BAZ1A protein, human; D000092468:Bromodomain Containing Proteins; C086468:ISWI protein; C081573:DNA modification methylase SssI; C084108:SMARCA4 protein, human; C409462:SMARCA5 protein, human