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Identification and Analysis of Native HAT Complexes
154
[8]
histone modifying enzymes
[8] Identification and Analysis of Native HAT Complexes By Stacey J. McMahon, Yannick Doyon, Jacques Coˆte´ , and Patrick A. Grant Acetylation of the amino-terminal tails of core histone proteins has been linked to many cellular processes, including cell cycle progression, DNA replication, chromatin assembly, and the regulation of gene expression.1 Therefore, characterization of histone acetyltransferases (HATs), the enzymes responsible for posttranslational acetylation of histones, is necessary to better understand the regulation of these processes. Two classes of HATs have been described: nuclear type A HATs, which mediate transcription related acetylation,2 and cytoplasmic type B HATs, which mediate histone acetylation related to subsequent chromatin assembly.1 Upon identification and purification of the first nuclear HAT from Tetrahymena, a homologue of the yeast transcriptional activator Gcn5, a direct link between histone acetylation and gene activation was established.2 It has since become apparent that substrate specificity is different between recombinant HAT enzymes and HATs as they typically exist in vivo in high molecular weight complexes.3 Therefore, it is necessary to purify native histone acetyltransferase complexes in order to fully understand the functions of the HAT enzymes themselves. Here, we will describe methods commonly used to purify and analyze native HAT complexes from the yeast Saccharomyces cerevisiae and human cells. These approaches have also been adapted for the purification of histone kinase and methytransferase complexes.4,5 Purification of Native Yeast HAT Complexes
The first method of purification relies on the binding of HATs to nickel agarose resin in the absence of any tags. It was found that numerous native yeast HAT complexes fortuitously bind to nickel.6 Whole cell extracts from yeast are prepared in the following manner. 1
S. Y. Roth, J. M. Denu, and C. D. Allis, Annu. Rev. Biochem. 70, 81 (2001). J. E. Brownell, J. Zhou, T. Ranalli, R. Kobayashi, D. G. Edmondson, S. Y. Roth, and C. D. Allis, Cell 84, 843 (1996). 3 P. A. Grant and S. L. Berger, Semin. Cell Dev. Biol. 10, 169 (1999). 4 B. D. Strahl, P. A. Grant, S. D. Briggs, Z. W. Sun, J. R. Bone, J. A. Caldwell, S. Mollah, R. G. Cook, J. Shabanowitz, D. F. Hunt, and C. D. Allis, Mol. Cell. Biol. 22, 1298 (2002). 2
METHODS IN ENZYMOLOGY, VOL. 377
Copyright 2004, Elsevier Inc. All rights reserved. 0076-6879/04 $35.00
[8]
histone modifying enzymes
[8] Identification and Analysis of Native HAT Complexes By Stacey J. McMahon, Yannick Doyon, Jacques Coˆte´ , and Patrick A. Grant Acetylation of the amino-terminal tails of core histone proteins has been linked to many cellular processes, including cell cycle progression, DNA replication, chromatin assembly, and the regulation of gene expression.1 Therefore, characterization of histone acetyltransferases (HATs), the enzymes responsible for posttranslational acetylation of histones, is necessary to better understand the regulation of these processes. Two classes of HATs have been described: nuclear type A HATs, which mediate transcription related acetylation,2 and cytoplasmic type B HATs, which mediate histone acetylation related to subsequent chromatin assembly.1 Upon identification and purification of the first nuclear HAT from Tetrahymena, a homologue of the yeast transcriptional activator Gcn5, a direct link between histone acetylation and gene activation was established.2 It has since become apparent that substrate specificity is different between recombinant HAT enzymes and HATs as they typically exist in vivo in high molecular weight complexes.3 Therefore, it is necessary to purify native histone acetyltransferase complexes in order to fully understand the functions of the HAT enzymes themselves. Here, we will describe methods commonly used to purify and analyze native HAT complexes from the yeast Saccharomyces cerevisiae and human cells. These approaches have also been adapted for the purification of histone kinase and methytransferase complexes.4,5 Purification of Native Yeast HAT Complexes
The first method of purification relies on the binding of HATs to nickel agarose resin in the absence of any tags. It was found that numerous native yeast HAT complexes fortuitously bind to nickel.6 Whole cell extracts from yeast are prepared in the following manner. 1
S. Y. Roth, J. M. Denu, and C. D. Allis, Annu. Rev. Biochem. 70, 81 (2001). J. E. Brownell, J. Zhou, T. Ranalli, R. Kobayashi, D. G. Edmondson, S. Y. Roth, and C. D. Allis, Cell 84, 843 (1996). 3 P. A. Grant and S. L. Berger, Semin. Cell Dev. Biol. 10, 169 (1999). 4 B. D. Strahl, P. A. Grant, S. D. Briggs, Z. W. Sun, J. R. Bone, J. A. Caldwell, S. Mollah, R. G. Cook, J. Shabanowitz, D. F. Hunt, and C. D. Allis, Mol. Cell. Biol. 22, 1298 (2002). 2
METHODS IN ENZYMOLOGY, VOL. 377
Copyright 2004, Elsevier Inc. All rights reserved. 0076-6879/04 $35.00