Abstract
Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V), at sites involved in RNA Polymerase (RNAP) II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG) transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase). As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination.
Highlights
Kinetoplastids are early-diverged protozoa that include the human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, which cause African sleeping sickness, Chagas disease, and leishmaniasis, respectively
The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter
We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J leads to readthrough transcription and the expression of downstream genes
Summary
Kinetoplastids are early-diverged protozoa that include the human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, which cause African sleeping sickness, Chagas disease, and leishmaniasis, respectively. The genomes of kinetoplastids are arranged into long gene clusters, or polycistronic transcription units (PTUs), which are transcribed by RNA polymerase (RNAP) II [1,2,3]. RNAP II transcription initiation and termination occurs at regions flanking PTUs called divergent strand switch regions (dSSRs) and convergent strand switch regions (cSSRs), respectively [4]. The arrangement of genes into PTUs has led to the assumption that transcription is an unregulated process in these eukaryotes and a model in which gene regulation occurs strictly post-transcriptionally [11, 12]. Specific chromatin marks have been characterized at sites of transcription initiation and termination, including histone variants and modified DNA base J, which could function to regulate polycistronic transcription and gene expression [13,14,15,16,17]
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