Abstract
Chromatin remodelling is involved in the transcriptional regulation of the RNA polymerase I transcribed variant surface glycoprotein (VSG) expression sites (ESs) of Trypanosoma brucei. We show that the T. brucei FACT complex contains the Pob3 and Spt16 subunits, and plays a key role in ES silencing. We see an inverse correlation between transcription and condensed chromatin, whereby FACT knockdown results in ES derepression and more open chromatin around silent ES promoters. Derepressed ESs show increased sensitivity to micrococcal nuclease (MNase) digestion, and a decrease in histones at silent ES promoters but not telomeres. In contrast, FACT knockdown results in more histones at the active ES, correlated with transcription shut-down. ES promoters are derepressed in cells stalled at the G2/M cell cycle stage after knockdown of FACT, but not in G2/M cells stalled after knockdown of cyclin 6. This argues that the observed ES derepression is a direct consequence of histone chaperone activity by FACT at the G2/M cell cycle stage which could affect transcription elongation, rather than an indirect consequence of a cell cycle checkpoint. These experiments highlight the role of the FACT complex in cell cycle-specific chromatin remodelling within VSG ESs.
Highlights
In eukaryotes gene expression is typically coordinated by complicated protein networks, which extend far beyond the scope of the basic transcription machinery
Chromatin remodelling is involved in the transcriptional regulation of the RNA polymerase I transcribed variant surface glycoprotein (VSG) expression sites (ESs) of Trypanosoma brucei
We show that the T. brucei FACT complex contains the Pob3 and Spt16 subunits, and plays a key role in ES silencing
Summary
In eukaryotes gene expression is typically coordinated by complicated protein networks, which extend far beyond the scope of the basic transcription machinery. Most T. brucei genes are present in very extensive polycistronic transcription units, which are constitutively transcribed by RNA polymerase II (Pol II) (Berriman et al, 2005). The bloodstream-form trypanosome expresses an essential protective VSG coat (Sheader et al, 2005) from a single copy VSG gene located in one of about 15 telomeric ES transcription units (Berriman et al, 2002; Hertz-Fowler et al, 2008). The active ES is transcribed by RNA polymerase I (Pol I) (Gunzl et al, 2003), which in eukaryotes normally exclusively transcribes rDNA. This unusual and highly regulated Pol I-mediated mono-allelic transcription of ESs in T. brucei is key for antigenic variation to work (Borst, 2002)
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