The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei.
The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.
Denninger
Viola
V
Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College, South Kensington, London SW72AZ, UK.
Fullbrook
Alexander
A
Bessat
Mohamed
M
Ersfeld
Klaus
K
Rudenko
Gloria
G
D000595:Amino Acid Sequence; D002453:Cell Cycle; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication; D016054:DNA, Protozoan / Q000096:biosynthesis; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D014643:Variant Surface Glycoproteins, Trypanosoma