Student Theses and Dissertations

Date of Award

2009

Document Type

Thesis

RU Laboratory

Cross George Laboratory

Keywords

Trypanosoma brucei, gene regulation, trans splicing efficiency, histone variants, RNA pol II transcription factors

Abstract

The protozoan parasite Trypanosoma brucei is one of the most divergent well-studied eukaryotes and many discoveries of general interest have been made in T. brucei. Atypical for a eukaryote, in T. brucei genes transcribed by RNA polymerase II (RNA pol II) are arranged in polycistronic transcription units (PTUs). mRNAs are separated post-transcriptionally by coupled splicing and polyadenylation reactions. During the splicing reaction a 39 nt ‘sliced-leader’ is added to every mRNA, a process termed trans splicing. The arrangement of generally unrelated genes in PTUs has lead to the assumption that little gene regulation occurs at the level of transcription initiation but that gene expression is regulated at the levels of mRNA maturation and stability. To better understand gene regulation in T. brucei, I performed a systematic analysis to correlate trans-splicing efficiency with specific DNA sequence motifs and observed large variations in trans-splicing efficiency depending on the DNA sequence upstream of the 3´ splice site. With one exception, no RNA pol II promoter motif has been identified in T. brucei, and how transcription is initiated remains an enigma. The second part of my thesis was based on the assumption that, given the apparent lack of RNA pol II promoter motifs, RNA pol II transcription start sites (TSS) are marked by distinct chromatin structures that facilitate recruitment of the transcription machinery. T. brucei has four histone variants: H2AZ, H2BV, H3V and H4V. Using ChIP-seq to examine the genome-wide distribution of chromatin components, I showed that H2AZ, H2BV, the K10-Ac form of H4, and the bromodomain factor BDF3 are significantly enriched at probable RNA pol II TSSs and used this mark to identify more than 60 previously unanticipated TSS candidates. Co-IP experiments with tagged H2A, H2AZ, H2B, and H2BV indicated that less histone H3 and histone H4 coimmunoprecipitated with variant histones than with core histones suggesting that variant nucleosomes are less stable than canonical nucleosomes. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable RNA pol II transcription termination sites. These findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general RNA pol II transcription factors have been elusive.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/426

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