Student Theses and Dissertations

Veena Mandava

Date of Award

2008

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Cross George Laboratory

Abstract

Trypanosoma brucei maintains an infection in its mammalian host by switching its surface antigen, thus evading host antibodies, in a process known as antigenic variation. Variant surface glycoproteins (VSG), the surface antigen, are expressed from genes located at as many as 20 Expression Sites (ES), which are present in subtelomeric regions of several chromosomes. How trypanosomes maintain monoallelic expression of VSG is a major question in trypanosome biology. Several theories have been proposed, including the presence of an Expression Site Body (a dedicated ‘transcription factory’), regulation of RNA elongation, and some kind of transcriptionally restrictive chromatin structure at inactive ES. In other organisms, the role of histone posttranslational modifications (PTMs) in influencing transcriptional states has been well-documented. To begin to understand the role of chromatin structure in the regulation of gene expression in trypanosomes, I developed a protocol for the purification of histones from mammalian-infective trypanosomes and identified PTMs using Edman degradation and mass spectrometry. I found that the N-termini of H4 and, possibly, H3 have a number of posttranslational modifications (PTMs), while H2A and H2B, in contrast, have relatively few. I also found a series of acetylated lysines at the C-terminus of H2A. Interestingly, I observed that alanine 1 of H2A, H2B, and H4 is monomethylated, a novel modification found in trypanosomatids. Next, I attempted to identify the targets of the putative histone deacetylase TbSIR2RP-1, a SIR2-related protein. Yeast SIR2 plays a key role in establishing heterochromatin at subtelomeric loci. TbSIR2RP-1 was chosen as a possible mediator of ES silencing, although this was later shown not to be the case. I showed that, unlike its yeast counterpart, TbSIR2RP-1 may not be an H4 deacetylase. Finally, I began to characterize H3 K4 methylation, which, in other organisms, is traditionally associated with transcriptionally active chromatin. I showed that nucleosomes containing the histone variant H2BV are enriched for H3 K4 methylation. I initially hypothesized that this is due to ubiquitination at a conserved lysine at the H2BV C-terminus, but find that this is not the case. Instead, I propose that replacement of major histones H2A/H2B with H2AZ/H2BV acts as a trigger to stimulate H3 K4 methylation, which represents an alternative pathway leading to H3 methylation, compared to what is observed in yeast.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Download

Included in

Life Sciences Commons

Share

COinS