Genetic Interaction Between Site-Specific Epigenetic Marks and Roles of H4v in Transcription Termination in Trypanosoma brucei.

Hee-Sook Kim

doi:10.3389/fcell.2021.744878

Abstract

In Trypanosoma brucei, genes are assembled in polycistronic transcription units (PTUs). Boundaries of PTUs are designated transcription start sites and transcription termination sites (TTSs). Messenger RNAs are generated by trans-splicing and polyadenylation of precursor RNAs, and regulatory information in the 3′ un-translated region (UTR), rather than promoter activity/sequence-specific transcription factors, controls mRNA levels. Given this peculiar genome structure, special strategies must be utilized to control transcription in T. brucei. TTSs are deposition sites for three non-essential chromatin factors—two of non-canonical histone variants (H3v and H4v) and a DNA modification (base J, which is a hydroxyl-glucosyl dT). This association generated the hypothesis that these three chromatin marks define a transcription termination site in T. brucei. Using a panel of null mutants lacking H3v, H4v, and base J, here I show that H4v is a major sign for transcription termination at TTSs. While having a secondary function at TTSs, H3v is important for monoallelic transcription of telomeric antigen genes. The simultaneous absence of both histone variants leads to proliferation and replication defects, which are exacerbated by the J absence, accompanied by accumulation of sub-G1 population. Thus, I propose that the coordinated actions of H3v, H4v, and J provide compensatory mechanisms for each other in chromatin organization, transcription, replication, and cell-cycle progression.

Highlights

Trypanosoma brucei is a parasitic protist that causes African sleeping sickness in humans and related diseases in animals, predominantly in sub-Saharan Africa
H3v J and J mutant cells exhibited transcription termination defects, these cells grew normally. These data suggest that successful transcription termination in T. brucei may require three non-essential chromatin marks (H3v, H4v, and base J) that coincide at termination sites (TTSs), which may functionally “signal” RNA polymerase II to stop
To avoid any obscurity caused by unknown events occurring during prolonged period of culturing after transfection and, to analyze the mutant phenotypes immediately after the knockout, I generated a conditional KO mutant for H3v H4v double KO, a H4v strain with a floxed H3v-Ty1 [conditional double KO (DKO)]

Summary

Introduction

Trypanosoma brucei is a parasitic protist that causes African sleeping sickness in humans and related diseases in animals, predominantly in sub-Saharan Africa. Disease transmission to mammals occurs by infected tsetse fly bites. To adapt in two different hosts, T. brucei undergoes stages of life cycle-specific differentiation. Trypanosomes proliferate as a procyclic form (PF) in the midgut and migrate to the fly’s salivary gland and differentiate into a non-proliferative metacyclic form. The infected fly injects the metacyclic form T. brucei into the mammalian host’s bloodstream. After differentiating into a bloodstream form (BF), trypanosomes proliferate in the host’s bloodstream and extracellular spaces.

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Conclusion