Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei

Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G₂/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G₂/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.     

Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei

We have studied the genes coding for the variant-specific surface antigen (VSA) in a series of seven trypanosome clones derived from AnTat 1.1: 1.1 leads to 1.3 leads to 1.6 leads to 1.16 leads to 1.1C leads to 1.3B leads to 1.18 These genes are all telomeric (1-5), and their surrounding, although sometimes similar, differs in each case. The length between these antigen genes and the corresponding DNA end appears to increase at each antigenic switch, with however occasional sharp size reductions, often linked to the involvement of the telomere in gene expression. This increase is due to a constant "growth" of the telomeres, at a rate of about 28 bp per day in at least four cases and probably linked to chromosome duplication. The telomere harbouring the transcribed VSA gene is growing slightly faster (about 36 bp per day), and it is the only one whose size reduction is progressive, leading to a terminal length heterogeneity within a clone. As a result, the active VSA gene is found in a population of telomeres which, as the trypanosomes divide, becomes increasingly heterogeneous, with however a preferred discrete size class about 1.4 kb smaller. The fact that the "active" telomere is the only one in a chromatin conformation highly sensitive to DNAaseI (1-4, 6), suggests that chromatin structure influences the rate and extent of both size increase and shortening of telomeres.     

Stable expression of biologically active recombinant bovine interleukin-4 in Trypanosoma brucei.

We have explored the potential of Trypanosoma brucei as a eukaryotic expression system. Procyclic forms, which correspond to an insect-adapted stage, can easily be cultured in vitro. The cells grow to densities approximately 10-fold greater than higher eukaryotic cells and are not infectious for mammals. An expression vector which can stably integrate into the genome was used to express high levels of recombinant bovine interleukin-4 (IL-4). Trypanosome-derived IL-4 is released into the medium and is biologically active. The recombinant protein down-regulates CD14 expression in human macrophages and inhibits NO production by stimulated bovine macrophages.     

The IsTat 1.3 VSG multigene family in Trypanosoma brucei: retention of the expression linked copy through multiple antigenic switches.

In the IsTaR 1 serodeme of T. brucei the 3 variant surface glycoprotein (VSG) gene family contains about 10 members, one of which has a telomeric location on a minichromosome. The expression linked copy (ELC) of the 3 VSG gene which occurs in an antigenic variant expressing the 3 VSG, also has a telomeric location but unlike the minichromosomal 3 VSG gene has restriction sites upstream from the 5' barren region. This ELC is retained on the same telomere in a subsequent variant that expresses a telomeric 7 VSG ELC and in relapse variants and procyclic forms derived from variant antigenic types (VATs) 3 and 7. The 7 ELC has a restriction map upstream from the 5' barren region that differs from, but is similar to, that of the 3 ELC. These data indicate that the 3 and 7 ELCs are on different telomeres when expressed.