Mechanisms of transcription factor integration at the c-myc promoter

Division Of Clinical Sciences - Nci

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Abstract

The Far UpStream Element Binding Protein (FUBP1) regulates MYC expression. Upon activation of the transcription of the MYC gene, torsional stress is dynamically pumped into the upstream regions of the MYC promoter. The FUSE region melts in response to this stress and binds FUBP1, which in turns activates TFIIH to increase output of the MYC promoter. as FUBP1 cycles on and off FUSE, high MYC expression is sustained. We hypothesize that to terminate the burst of MYC transcription, the pulse of MYC transcription, FUBP1 and FUSE activity must first be ended. If a mechanism existed to drain the torsional stress in the FUSE-region, the melted bubble there would collapse, evicting FUBP1 and preventing its re-binding. We have discovered a type 1 topoisomerase activity in highly affinity-purified preparations of FUBP1. Upon further purification we found a small fraction of the FUBP1 was chromatographically separable from the bulk of the protein. This small fraction contained all of the topoisomerase activity (but was nevertheless immunologically and by mass spec derived from FUBP1). We are trying to define the active site of the protein. Recent experiments indicate the importance of cysteine residues for topoisomerase activity. If so, this will help us to define conditions and strategies to control fubp1 activity, and thereby control MYC. We speculate that this protein acts to terminate bursts of MYC transcription by shutting down FUSE. If this is true it will provide new insights and therapeutic strategies to control MYC expression. We aim to reconstitute the transcription of the MYC promoter in the in vitro transcription system being developed by Dr. Brian Lewis that conduct reactions in phase separated biomolecular condensates that potentially will allow us to study in detail the mechanism of action of FuBP1 and other transcription regulators.