Biology and Biomedical Sciences: Molecular Cell Biology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Stephen M Beverley
Leishmania spp. and other trypanosomatid protozoa use a highly unusual mechanism to generate functional messenger RNAs (mRNAs) in which protein-coding genes are transcribed polycistronically. Here, transcription initiates primarily in divergent strand switch regions (dSSRs), where two polycistronic gene clusters are oriented head-to-head. These regions lack all known eukaryotic cis-regulatory elements, and it is not known how genetic and epigenetic factors cooperate to define dSSRs as regions of productive initiation. To quantitatively identify regulatory elements and to study the contribution of epigenetic factors to dSSR function, we combined genome-wide studies of chromatin structure with a focused interrogation of a single dSSR using a novel integrated bidirectional, dual-luciferase reporter. Chromatin-based studies demonstrated that Leishmania lack well-positioned nuclease-hypersensitive sites associated with promoters in other eukaryotes. Rather, nuclease-hypersensitive sites are positioned heterogeneously across broad regions associated with epigenetic marks indicative of active transcription, suggesting that transcription initiation events occur promiscuously within regions associated with a transcriptionally-permissive epigenetic state. Our studies using the bidirectional reporter validate these observations and strongly suggest that Leishmania do not require cis-regulatory elements for efficient bidirectional transcription initiating in dSSRs, as a large region of the dSSR can be replaced with unrelated sequences without altering bidirectional reporter gene expression. In addition to these genetic studies we also focused on epigenetic determinants of transcriptional activity in Leishmania, with respect to both transcription initiation and transcription termination. We showed that the histone variants H2A.Z and H2B.V, which are associated with transcriptionally permissive regions in T. brucei, are essential in L. major, while the transcription termination-associated histone variant H3.V is not. Interestingly, unlike Leishmania lacking the DNA modification base J, H3.V-null L. major shows no defects in transcription termination. Although the study of essential genes in Leishmania is challenging at this time, we present preliminary data describing elements of inducible gene expression systems which may improve our ability to study essential genes. Together, the data in this thesis show that transcription of protein-coding genes is primarily determined epigenetically, and suggest that chromatin-related processes may be an attractive target for therapeutic intervention.
Anderson, Britta, "Genetic and Epigenetic Determinants of Transcription in the Divergent Eukaryote Leishmania major" (2014). All Theses and Dissertations (ETDs). 1279.