Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei

摘要

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. Author Summary Trypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping sickness in humans. The genome of T . brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. Because of this genome arrangement, it is thought that all gene regulation in T . brucei occurs after transcription at the level of RNA (processing, stability, and translation). 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 read-through transcription and the expression of downstream genes. We now find that H3.V also promotes termination prior to the end of gene clusters, thus regulating the transcription of specific genes. Additionally, H3.V inhibits transcription of siRNA producing loci. Our data suggest H3.V and base J are utilized for regulating gene expression via terminating transcription within polycistronic gene arrays and regulating the synthesis of siRNAs in trypanosomes. These findings significantly expand our understanding of epigenetic regulatory mechanisms underlying transcription termination in eukaryotes, including divergent organisms that utilize polycistronic transcription, providing the first example of a histone variant that promotes transcription termination.