Trypanosoma bruceiOrc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching

Abstract

Summary Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host’s immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homolog has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in-situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…How stabilization might occur remains uncertain (103), in part because the trigger and timing of transcriptional VSG switching is unknown. DNA replication and segregation may provide a window for transcriptional change, which would be consistent with observations that RNAi targeting of factors involved in nuclear replication and chromatid cohesion functions can elevate levels of silent VSG expression and switching (104)(105)(106)(107). However, whether directed or random events can precipitate a transcriptional switch remains unknown.…”

“…5) have been limited to understanding the activation of intact VSGs (106,108,110,114,117,(127)(128)(129). This stems from the fact that these studies have used in vivo or in vitro selection strategies that detect the most frequently activated VSGs; the lack of detectable mosaic VSG formation in these studies may suggest that SGC is less efficient than intact VSG conversion, though a mechanistic switch during an infection cannot be currently ruled out.…”

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…The precise underlying molecular mechanism is still not understood. Although several molecular players have been shown to be involved (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), depletion of any of these factors had only a moderate effect on the repression of silent VSG genes. Therefore, either a master regulator is still to be discovered or, alternatively, the process is controlled by multiple collaborative factors ensuring a cumulative tight lock.…”

“…Furthermore, the level at which the control takes place is unknown: Whether the transcription control takes place at the initiation or elongation level is debated. Data reporting low levels of transcripts from silent ESs upon RNAi-mediated knockdown of various factors (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) cannot resolve this issue, because the data are compatible with both models. Previous experiments pointed to mixed conclusions.…”

“…The mechanism controlling the selective activity of a single ES at a time is still unknown, although several factors have been found to be involved, such as differential chromatin/histone acetylation and methylation between active and inactive ESs (6-8); nucleosome depletion in the active ES (9,10); and the influence of various negative transcription modulators such as a histone methylase, a high-mobility group protein, histone H1, NLP (a nucleoplasm-like protein), a FACT subunit (a chromatin remodeler), a SWI/SNF (a chromatin remodeling factor), ORC1 and MCM-BP (proteins involved in the initiation of DNA replication), RAP1 (a telomere-binding protein), histone chaperones, or a lamin-like protein (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). However, no master ES regulator has been identified so far.…”