Measurement of histone acetyltransferase and histone deacetylase activities and kinetics of histone acetylation.
Dynamic histone acetylation has a role in chromatin remodeling and in the regulation of transcription. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) catalyze reversible histone acetylation. HATs and HDACs exist as multiprotein complexes that have coactivator and corepressor activities, respectively. The steady-state level of acetylation at a chromatin site is determined by the local net activities of these enzymes. Here we describe methods to isolate different subcellular fractions (cytosol, nuclei, tightly bound nuclear, loosely bound nuclear, immunoprecipitated multiprotein complexes, and nuclear matrix) to determine the subcellular distribution of HAT and HDAC activities. Procedures to assay the activities of these enzymes and to measure the kinetics of histone acetylation and deacetylation are presented.
Sun
Jian-Min
JM
Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Man., Winnipeg, Canada R3E 0V9.
Spencer
Virginia A
VA
Chen
Hou Yu
HY
Li
Lin
L
Davie
James R
JR
D000107:Acetylation; D000123:Acetyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D002645:Chickens; D002845:Chromatography / Q000379:methods; D019281:Dimerization; D017886:Durapatite; D004912:Erythrocytes / Q000201:enzymology; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases / Q000302:isolation & purification* / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals
D006657:Histones; D017886:Durapatite; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases