Analysis of transcription factor-mediated remodeling of nucleosomal arrays in a purified system.
An early step in a pathway leading to transcriptional initiation involves the rearrangement of chromatin at gene regulatory sequences. To study this process, we have developed a biochemical system analyzing the interactions between chromatin templates composed of arrays of positioned nucleosomes and sequence-specific transcriptional activators. Here, a procedure is presented for the assembly of nucleosomal arrays on DNA fragments containing synthetic and natural gene sequences inserted within tandem repeats of sea urchin 5S rDNA. We also provide methods for the use of these templates in transcription factor-binding assays, as well as experimental data illustrating the efficacy of such analyses to uncover mechanisms directing factor-mediated nucleosome remodeling.
Steger
D J
DJ
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4500, USA.
Owen-Hughes
T
T
John
S
S
Workman
J L
JL
D002474:Cell-Free System; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D009707:Nucleosomes; D012341:RNA, Ribosomal, 5S; D014157:Transcription Factors