Using quantitative imaging microscopy to define the target substrate specificities of histone post-translational-modifying enzymes.
Until recently, identifying the specificities of enzymes that post-translationally modify core histones was performed in vitro using synthetic peptides, purified mononucleosomes or short nucleosome arrays. Unfortunately, the variable results obtained for identical enzymes are often dependent on the in vitro conditions employed. These results are consistent with the conclusion that the manner in which histone tails are presented to the modifying enzymes dramatically affects specificity. Because traditional in vitro biochemical approaches do not accurately recapitulate higher-order chromatin structure or consider the influences that additional chromatin binding proteins may have on determining the specificity of modifying enzymes, the development of new and innovative approaches is warranted. Here, we describe a novel in situ microscopy approach that accurately assesses enzyme substrate specificities through single cell measurements performed under physiologically relevant conditions. This approach couples the spatial resolving power of microscopy with robust statistical analyses to determine the substrate specificities of transiently expressed enzymes using histone modification- and residue-specific antibodies. This methodology can also be applied to measuring changes in the abundance of histone modifications as cells traverse the cell cycle.
McManus
Kirk J
KJ
Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alta., Canada T6G 1Z2.
Hendzel
Michael J
MJ
D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D050882:CREB-Binding Protein; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D019411:Clinical Laboratory Techniques; D003627:Data Interpretation, Statistical; D004798:Enzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D051548:Histone Acetyltransferases; D006657:Histones / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008856:Microscopy, Fluorescence / Q000379:methods; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D000906:Antibodies; D050882:CREB-Binding Protein; D018797:Cell Cycle Proteins; D004798:Enzymes; D005456:Fluorescent Dyes; D051548:Histone Acetyltransferases; D006657:Histones; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C494363:CREBBP protein, human