Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips.
Chromatin-remodeling complexes alter chromatin structure to facilitate, or in some cases repress, gene expression. Recent studies have suggested two potential pathways by which such regulation might occur. In the first, the remodeling complex repositions nucleosomes along DNA to open or occlude regulatory sites. In the second, the remodeling complex creates an altered dimeric form of the nucleosome that has altered accessibility to transcription factors. The extent of translational repositioning, the structure of the remodeled dimer, and the presence of dimers on remodeled polynucleosomes have been difficult to gauge by biochemical assays. To address these questions, ultrahigh-resolution carbon nanotube tip atomic force microscopy was used to examine the products of remodeling reactions carried out by the human SWI/SNF (hSWI/SNF) complex. We found that mononucleosome remodeling by hSWI/SNF resulted in a dimer of mononucleosomes in which approximately 60 bp of DNA is more weakly bound than in control nucleosomes. Arrays of evenly spaced nucleosomes that were positioned by 5S rRNA gene sequences were disorganized by hSWI/SNF, and this resulted in long stretches of bare DNA, as well as clusters of nucleosomes. The formation of structurally altered nucleosomes on the array is suggested by a significant increase in the fraction of closely abutting nucleosome pairs and by a general destabilization of nucleosomes on the array. These results suggest that both the repositioning and structural alteration of nucleosomes are important aspects of hSWI/SNF action on polynucleosomes.
Schnitzler
G R
GR
Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA. gavin.schnitzler@tufts.edu
Cheung
C L
CL
Hafner
J H
JH
Saurin
A J
AJ
Kingston
R E
RE
Lieber
C M
CM
D002244:Carbon / Q000737:chemistry*; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D008861:Microsomes / Q000378:metabolism; D008957:Models, Genetic; D009687:Nuclear Proteins; D009707:Nucleosomes / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes; D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; C499336:SMARCA1 protein, human; C084011:SMARCA2 protein, human; D014157:Transcription Factors; D002244:Carbon; D004247:DNA; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; C084108:SMARCA4 protein, human; D004265:DNA Helicases