Epigenetic Regulation of Polymerase II Transcription Initiation in Trypanosoma cruzi: Modulation of Nucleosome Abundance, Histone Modification, and Polymerase Occupancy by O-Linked Thymine DNA Glucosylation

Abstract

Very little is understood regarding how transcription is initiated/regulated in the early-diverging eukaryote Trypanosoma cruzi. Unusually for a eukaryote, genes transcribed by RNA polymerase (Pol) II in T. cruzi are arranged in polycistronic transcription units (PTUs). On the basis of this gene organization, it was previously thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. We recently localized a novel glucosylated thymine DNA base, called base J, to potential promoter regions of PTUs throughout the trypanosome genome. Loss of base J, following the deletion of JBP1, a thymidine hydroxylase involved with synthesis, led to a global increase in the Pol II transcription rate and gene expression. In order to determine the mechanism by which base J regulates transcription, we have characterized changes in chromatin structure and Pol II recruitment to promoter regions following the loss of base J. The loss of base J coincides with a decrease in nucleosome abundance, increased histone H3/H4 acetylation, and increased Pol II occupancy at promoter regions, including the well-characterized spliced leader RNA gene promoter. These studies present the first direct evidence for epigenetic regulation of Pol II transcription initiation via DNA modification and chromatin structure in kinetoplastids as well as provide a mechanism for regulation of trypanosome gene expression via the novel hypermodified base J.Trypanosoma cruzi is a major human parasitic pathogen with a complex life cycle, alternating between an insect vector and mammalian hosts (42). In both, T. cruzi undergoes differential morphological and functional changes that require rapid and selective changes in gene expression profile. Unlike other eukaryotes, the genes in T. cruzi are arranged in large polycistronic bidirectional gene clusters (34). These polycistronic transcription units (PTUs) are transcribed by RNA polymerase (Pol) II to yield polycistronic pre-mRNAs that are then processed into mature mRNAs by trans-splicing and polyadenylation. Given this genome arrangement and the apparent lack of a functional relationship of genes within a PTU, the regulation of trypanosome gene expression was thought to occur primarily via differential mRNA decay or other posttranscriptional mechanisms (5). Until very recently, there was no evidence for regulation of gene expression at the level of Pol II transcription in kinetoplastids (11). Furthermore, very little is understood about the DNA sequences and proteins involved in transcription initiation and termination in kinetoplastids. With the exception of the spliced-leader RNA promoter, Pol II promoters and associated factors have not been identified or functionally characterized. While genome localization studies suggest that histone modifications and histone variants are involved in transcription initiation and termination in trypanosomes, direct evidence from functional studies is lacking (34, 39). Thus, how Pol II transcription is initiated/regulated, including the rol...

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

“…Interestingly, 5-formylcytosine and 5-carboxycytosine can significantly reduce the kinetics of yeast RNA polymerase II transcription, suggesting that these modifications play a role in splicing and termination (46) intriguing because the studies demonstrating the role of base J in Pol II transcription were performed by halting hmU synthesis via altering JBP function (5)(6)(7). Future experiments utilizing the JGT KO cell line will allow us to address the specific role of hmU, fU, and base J in regulating Pol II kinetics and trypanosomatid gene expression.…”

JBP1 and JBP2 Proteins Are Fe2+/2-Oxoglutarate-dependent Dioxygenases Regulating Hydroxylation of Thymidine Residues in Trypanosome DNA

“…The loss of base J at transcription start sites coincides with a decrease in nucleosome abundance, increased histone acetylation, and increased Pol II occupancy at promoter regions (3,4). This increase in Pol II recruitment correlates with an increased rate of transcription initiation and changes in gene expression (3). These studies indicate the importance of epigenetic regulation of Pol II transcription via DNA modification and chromatin structure in kinetoplastids as well as provide a mechanism for J regulation of trypanosome gene expression.…”

“…We have previously demonstrated that reduced J levels in T. cruzi results in increased Pol II recruitment to promoter regions and an increase in the rate of transcription initiation, which in turn causes global changes in gene expression (3,4). Notably, surface proteins involved in parasite pathogenesis (including members of the trans-sialidase and mucin gene family) are significantly affected during reduced J levels.…”

“…Anti-J ChIP analysis indicated that whereas the remaining J in the T. cruzi JBP1 KO is located in telomeric DNA, base J is lost from the divergent and convergent strand-switch regions involved in Pol II initiation and termination, respectively. The loss of base J at transcription start sites coincides with a decrease in nucleosome abundance, increased histone acetylation, and increased Pol II occupancy at promoter regions (3,4). This increase in Pol II recruitment correlates with an increased rate of transcription initiation and changes in gene expression (3).…”

“…However, more recently, we localized a minor fraction of J to chromosome-internal regions coinciding with RNA polymerase II (Pol II) transcription initiation and termination sites (2). Loss of base J synthesis at these chromosome-internal regions in T. cruzi led to increased Pol II transcription initiation and corresponding changes in gene expression and parasite virulence (3,4). Thus, base J represents a novel epigenetic modification of kinetoplastid DNA involved in regulating gene expression.…”