SEATTLE BIOMEDICAL RESEARCH INSTITUTE
Despite more than 20 years of molecular investigation, little is currently known about the essential process of the transcription of protein-coding genes in Leishmania and other trypanosomatids, other than that it differs from that in other eukaryotes because mature mRNAs are generated by trans-splicing of large polycistronic primary transcripts. Moreover, there is no evidence for the transcriptional regulation of gene expression typically seen in other organisms. Previous work from our laboratory has shown that transcription initiates at only a small number of sites between large (50-500 kb) divergent clusters of adjacent protein-coding genes on the same DNA strand and proceeds bi-directionally before termination at the junction between convergent gene clusters. However, the promoter elements involved in transcription initiation remain a mystery. In other eukaryotes, histone modification and chromatin re-modeling are important for activation of transcription, and recent experiments suggest that this may also be the case in trypanosomatids. This has led us to hypothesize that the strand-switch regions between divergent PGCs recruit chromatin-remodeling complexes which modify histones and alter the local chromatin structure in these regions to allow access by the RNA polymerase II pre-initiation complex, which then initiates transcription in both directions. In this proposal, we will test this hypothesis and build on our current work to further characterize the chromatin-modifying enzymes that are involved in transcription initiation.