Quantitative analysis of CBP- and P300-induced histone acetylations in vivo using native chromatin.
In vivo, histone tails are involved in numerous interactions, including those with DNA, adjacent histones, and other, nonhistone proteins. The amino termini are also the substrates for a number of enzymes, including histone acetyltransferases (HATs), histone deacetylases, and histone methyltransferases. Traditional biochemical approaches defining the substrate specificity profiles of HATs have been performed using purified histone tails, recombinant histones, or purified mononucleosomes as substrates. It is clear that the in vivo presentation of the substrate cannot be accurately represented by using these in vitro approaches. Because of the difficulty in translating in vitro results into in vivo situations, we developed a novel single-cell HAT assay that provides quantitative measurements of endogenous HAT activity. The HAT assay is performed under in vivo conditions by using the native chromatin structure as the physiological substrate. The assay combines the spatial resolving power of laser scanning confocal microscopy with simple statistical analyses to characterize CREB binding protein (CBP)- and P300-induced changes in global histone acetylation levels at specific lysine residues. Here we show that CBP and P300 exhibit unique substrate specificity profiles, consistent with the developmental and functional differences between the two HATs.
McManus
Kirk J
KJ
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.
Hendzel
Michael J
MJ
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D050882:CREB-Binding Protein; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D050881:E1A-Associated p300 Protein; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D002843:Chromatin; D006657:Histones; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D000123:Acetyltransferases; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases; D008239:Lysine