SWI/SNF complexes and facilitation of TATA binding protein:nucleosome interactions.
It has become increasingly apparent that eukaryotic cells possess machinery that modifies chromatin structure and that this machinery contributes to the regulation of gene expression. Identification of factors that alter chromatin structure has made possible biochemical analyses that have begun to define what structural changes each factor can cause as well as what consequences these changes have on transcription factor function. Here, a protocol that has facilitated study of energy-dependent chromatin remodeling complexes containing SWI/SNF proteins is described. Rotationally phased mononucleosome particles were assembled in vitro and used to demonstrate that human SWI/SNF complexes and the yeast RNA polymerase II holoenzyme, which contains yeast SWI/SNF proteins, can directly alter nucleosome structure in an ATP-dependent manner. A functional consequence of this nucleosome disruption is that the pol II general transcription factor, TATA binding protein (TBP), which cannot bind to unaltered nucleosomal DNA, can bind to its site on the altered nucleosome. This experimental system has been invaluable for characterization of both nucleosome alteration and facilitated transcription factor binding mediated by SWI/SNF complexes. These procedures should also be useful to examine other factors that interact with or structurally affect nucleosome particles.
Imbalzano
A N
AN
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655, USA.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D004247:DNA / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins; D005990:Glycerol; D006657:Histones / Q000302:isolation & purification; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006657:Histones; D009707:Nucleosomes; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C089969:snf protein, Drosophila; D000255:Adenosine Triphosphate; D004247:DNA; D012319:RNA Polymerase II; D003850:Deoxyribonuclease I; D005990:Glycerol