The control of gene expression is vital to development, homeostasis, and signal transduction. In eukaryotes, DNA packaging into chromatin generally represses transcription. Thus, transcriptional activation often requires chromatin remodeling machinery, of which the two main classes are the histone acetyltransferases and several types of ATPases that disrupt or relocate nucleosomes. Chromatin remodeling by ATPases is regulated by and is essential to the cell cycle, and histone acetyltransferases are targeted by viral oncoproteins. The molecular mechanisms of remodeling are unclear, but a common aspect is that each class releases negative supercoiling from nucleosomes. In the context of chromatin, this is expected to generate substantial strain throughout the local topological domain. It was recently shown that the TATA box-binding protein (TBP), central to Pol II transcription initiation as part of the TFIID assembly, induces negative supercoiling upon DNA cyclization of short restriction fragments to give minicircies. This was explained by invoking a flattened, unwound form of the TBP-DNA complex. Combining these observations suggests a possible indirect long-range communication mechanism between the remodeling machinery and TBP/TFIID: TBP is expected to bind with increased affinity to remodeled chromatin, because the high free energy cost of local topological unwinding induced by remodeling can be absorbed by untwisting within the TBP-DNA complex. The effect should be transient, which is a biologically appealing mechanism for selectively enhancing transcription of newly-remodeled chromatin. The current proposal will critically evaluate the model and test other implications of previous work as follows: 1) The DNA length-dependence of topological changes induced by TBP, TFIID, TBP-TFIIA and other transcription factor assemblies upon DNA cyclization will be used to assess the geometry, flexibility, and stability of the putative flattened form. Well-established Monte Carlo simulation methods will be extended to these systems. 2) Quantitative footprinting experiments on TBP binding to minicircle DNA will be used to test the effect of pre-bending and pre-supercoiling on the pathway for TBP binding to DNA. 3) TBP mutants and TBP-related factors will be used to assess the importance of phenylalanine stirrups to TBP binding and topology, using DNA cyclization and/or minicircle binding assays. Several TBP-related factors are important in transcription, but their DNA binding properties have been difficult to study. It is possible that they are obligate DNA unwinding proteins. 4) A nucleosome positioned on the 5S gene in TATA-containing DNA minicircles will be used to test the remodeling-binding connection directly, in vitro. Long-range topological communication between the nucleosome and TBP is predicted to lead to repression of TBP binding. Remodeling the nucleosome with recombinant ISWI ATPase or purified yeast SWI/SNF should then potentiate TBP binding.
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