Discovery of Enzymatic Targets of Transcriptional Activators via in Vivo Covalent Chemical Capture.
The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of in vivo covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators.
Amanda
A
Dugan
Chinmay Y
CY
Majmudar
Rachel
R
Pricer
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California 92037, United States.
Sherry
S
Niessen
Jody K
JK
Lancia
Hugo Yik-Hong
HY
Fung
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California 92037, United States.
Benjamin F
BF
Cravatt
Anna K
AK
Mapp
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors