Nucleoprotein gel assays for nucleosome positioning and mobility.
Recent recognition of the sensitivity of polyacrylamide gel electrophoresis to macromolecular conformation has provided a source of new applications. In chromatin research, nucleoprotein gel electrophoresis can yield a direct and visual estimate of the number and relative abundance of different positions adopted by the core histone octamer on DNA, as well as their locations relative to the middle of the DNA fragment. It is the only technique available for the fractionation of such nucleosome positioning isomers and leaves them intact. Thus this simple method constitutes a powerful tool to analyze and manipulate populations of variously positioned nucleosomes in their native state. Complementing conventional invasive enzymatic procedures that rely on the analysis of cutting patterns on nucleosomal DNA, these procedures are now revealing that histone octamers can reconstitute to a number of discrete, often overlapping, locations on most DNA sequences. Further capitalizing on these advantages of nucleoprotein gel analysis, the development of the technique into a two-dimensional assay has permitted a rare view at the dynamics of nucleosome positioning. Nucleosomes can redistribute between possible positions on DNA, with the distribution patterns of nucleosomes along the DNA being in dynamic equilibrium at 37 degrees in relatively low ionic strength conditions. This mobility of nucleosomes on DNA means that possible positions of nucleosomes can be defined precisely but that the actual locations of the nucleosomes are dynamic. It provides a compelling argument that a nucleosome position should be regarded as a probability rather than a static factor type of binding. This supports a more dynamic view of the nucleosomal organization, which seems more in accordance with the dynamic nature of gene expression. In providing the flexibility for adaptation, multiple positioning and nucleosome mobility could constitute essential ingredients of the mechanisms by which chromatin participates in gene regulation.
Pennings
S
S
Department of Biomedical Sciences, University of Edinburgh, Scotland, United Kingdom.
D000107:Acetylation; D000818:Animals; D020029:Base Pairing; D002458:Cell Fractionation / Q000379:methods; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005990:Glycerol; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification*; D007202:Indicators and Reagents; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000737:chemistry*; D009707:Nucleosomes / Q000502:physiology / Q000648:ultrastructure*; D011487:Protein Conformation; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D012680:Sensitivity and Specificity
D002868:Chromosomal Proteins, Non-Histone; D004275:DNA, Ribosomal; D006657:Histones; D007202:Indicators and Reagents; D009698:Nucleoproteins; D009707:Nucleosomes; D012341:RNA, Ribosomal, 5S; D005990:Glycerol