Transcriptional control and chromatin
structure in African trypanosomes

We are trying to understand how VSG expression sites are turned on and off in a mutually exclusive fashion in bloodstream form T. brucei. We are attempting to identify genes involved in VSG expression site silencing and control.

Role of TbISWI in VSG expression site silencing

We have discovered the first gene shown to play a role in downregulation of VSG expression sites in T. brucei (TbISWI). TbISWI is a trypanosome member of the ISWI family of chromatin remodeling proteins, and plays a role in VSG expression site silencing in both bloodstream and insect form trypanosomes.

 

blocking


Opposite left: Blocking synthesis of TbISWI using RNAi leads to derepression of silent VSG expression sites as monitored using flow cytometry of a GFP gene inserted into a silent VSG expression site. At the bottom are FACs traces of GFP derepression measured in the FITC channel after the induction of TbISWI RNAi for 80 hours.
From Hughes et al. (2007) EMBO J 26: 2400-2410.

 

 

 



fusion







Opposite right: TbISWI-GFP fusion protein located in the nucleus of trypanosomes at different stages in the cell cycle. The number of nuclei (N) or kinetoplasts (K) are indicated.
From Hughes et al. (2007) EMBO J 26: 2400-2410.

 

 

 



ISWI proteins are typically components of ISWI chromatin remodeling complexes, which can have different roles within the cell depending on which proteins ISWI is partnered up with. We would now like to determine the partners of TbISWI in order to get a better understanding of TbISWI complexes in T. brucei. We would like to dissect the different roles that TbISWI complexes can play in chromatin remodeling and regulation of gene expression in African trypanosomes.


Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes

The role that chromatin plays in VSG expression site control has been controversial, and it has not always been considered important. This issue has recently been reinvestigated with new experimental techniques. We and the Cross laboratory (Rockefeller University) have shown that active VSG expression sites (ES)s are depleted of nucleosomes compared with silent ones. This was shown using chromatin immunoprecipitation (ChIP) with antibodies against different trypanosome histones as well as fractionation of micrococcal nuclease digested trypanosome chromatin.

Therefore, in contrast to earlier views we now think that nucleosome positioning is involved in the monoallelic control of VSG ESs. This may provide a level of epigenetic regulation allowing bloodstream form trypanosomes to effectively pass on the transcriptional state of active and silent ESs to daughter cells.

We would now like to know more about how this nucleosome repositioning occurs during VSG switching. We would also like to identify protein complexes which could be involved in this chromatin remodeling.

vsg expression


Above: Schematics of T. brucei variant HNI(221+) expressing the VSG221 VSG expression site or T. brucei HNI(VO2+) expressing the VSGVO2 expression site. Primer pairs are indicated with letters. Chromatin immunoprecipitation (ChIP) experiments using an anti-Histone H4 antibody indicate that the active VSG expression site is relatively depleted of histone H4 compared with the silent VSG expression sites.
From Stanne and Rudenko (2010) Euk. Cell 9: 136-147.