Application of a fluorescent histone acetyltransferase assay to probe the substrate specificity of the human p300/CBP-associated factor.
Histone N-acetyltransferases (HATs) are a group of enzymes which acetylate specific lysine residues in the N-terminal tails of nucleosomal histones to promote transcriptional activation. Recent structural and enzymatic work on the GCN5/PCAF HAT family has elucidated the structure of their catalytic domain and mechanism of histone acetylation. However, the substrate specificity of these enzymes has not been quantitatively investigated. Utilizing a novel microplate fluorescent HAT assay which detects the enzymatic production of coenzyme A (CoA), we have compared the activities of the HAT domains of human PCAF and its GCN5 homologue from yeast and Tetrahymena and found that they have similar kinetic parameters. PCAF was further assayed with a series of different length histone H3 peptide substrates, which revealed that the determinants for substrate recognition lie within a 19-residue sequence. Finally, we evaluated the acetylation of three putative PCAF substrates, histones H3 and H4 and the transcription factor p53, and have determined that histone H3 is significantly preferred over the histone H4 and p53 substrates. Taken together, the fluorescent acetyltransferase assay presented here should be widely applicable to other HAT enzymes, and the results obtained with PCAF demonstrate a strong substrate preference for the N-terminal residues of histone H3.
Trievel
R C
RC
Department of Biochemistry and Biophysics, Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Li
F Y
FY
Marmorstein
R
R
D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D050882:CREB-Binding Protein; D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes / Q000737:chemistry*; D051548:Histone Acetyltransferases; D006657:Histones / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D050882:CREB-Binding Protein; D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes; D051548:Histone Acetyltransferases; D006657:Histones; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C494363:CREBBP protein, human; C501179:KAT2A protein, human