Procyclic gene expression

VSGs and expression sites

VSG regulation of bloodstream form

Differentiation from bloodstream to procyclic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Trypanosomal procyclic cell, taken from the Simpson Lab at UCLA.

 

 

Snapshots of the top and side views of a variant surface glycoprotein molecule from the 3D structure from Protein Databank.

High-level electron microscope magnification of the surface coat of T. brucei taken from the Rockefeller University Laboratory of Medical Parasitology website.

 

 

 

 

Bloodstream form of the trypanosome from Hill Lab at UCLA.

 

 

 

 

 

 

Trypanosomal stumpy form taken from the University of Manchester School of Biological Sciences.

 

Gene Expression in Trypanosoma brucei

Image taken from the University of Manchester
School of Biological Sciences.

Protein-coding genes lack class II introns and contain tandem repeats of either the same open reading frame (ORF) or unrelated sequences. The transcripts of the protein-coding genes are polycistronic. Between the protein-coding genes are specific intergenic sequences between the mRNA genes. Individual genes that belong to the same unit can show differing expression patterns (VanHamme & Pays, 1995).

The unusual RNA polymerases (pols) of trypanosomes are of three different types. Two of the RNA pols are transcribed from slightly different pol II genes (VanHamme & Pays, 1995). All active genes seem to be associated with pol I-type promoters, and no pol II-type promoters have been identified (Ersfeld, et al., 1999). Trypanosomes have novel variant surface glycoprotein (VSG) and procyclin promoters that are reminiscent of eukaryotic ribosomal promoters. Interestingly, the procyclin promoters are required for DNA replication (VanHamme & Pays, 1995).

Genes are generally regulated using posttranscriptional control. One method used by trypanosomes is the unique RNA editing mechanism in mitochondria. The expression of genes is dependent on the life cycle. For instance, there is a change in the levels of mitochondrial proteins due to regulation of both mitochondrial and nuclear gene expression. In the bloodstream slender form the mitochondria lack cristae and do not use cytochrome-mediated respiration, while in the procyclic form the mitochondria is larger, contains cristae, and uses oxidative metabolism (VanHamme & Pays, 1995).

 

Gene Expression Specific to the Procyclic Form

The procyclic acidic repetitive proteins (PARPs), PARP A and PARP B are transcribed from two unlinked loci. These genes are short, polycistronic, and nontelomeric. The procyclic genes are regulated by temperature and the same factors that block elongation in the regulation of VSG expression. During the procyclic stage, the presence of a 3’-UTR causes an increased amount of reporter mRNA through posttranscriptional regulation (VanHamme & Pays, 1995).

 

Variant surface glycoproteins and VSG expression sites

Trypanosomes evade immune response by altering their surface antigen, a protein homodimer called the variant surface glycoprotein (VSG). VSG molecules are attached to the plasma membrane with glycosol phosphatidylinositol (GPI) anchors. VSG expression only occurs in bloodstream and metacyclic stages of the life cycle, and most of the VSG genes are only expressed in the bloodstream form. There are on the order of 10^7 VSG molecules per bloodstream and metacyclic cell.

Activated VSG genes are contained within 45-60 kb polycistronic VSG expression sites (ESs). There are approximately 20 of VSG ESs in the entire trypanosomal genome. VSG ESs are located in the telomeric regions of intermediate and megabase chromosomes, 5-10 kb from the chromosome end. Only one VSG ESs is expressed at a time (VanHamme & Pays, 1995; VanHamme, et al., 2001).

Bloodstream VSG ESs (BESs) are large polycistronic arrays that contain several expression site-associated genes (ESAGs). BESs contain a VSG gene surrounded by a 3’ region of telomeric repeats (TTAGGG), a 5’ region of noncoding 50 bp repeats, and a few regions of 70 bp repeats among the ESAGs (see fig. 1). These barren repetitive regions behave as recombinatorial hot spots, which, as you will see, is important for the regulation of VSG genes in BESs (VanHamme & Pays, 1995). The T.b. rhodesiense BES does not contain several of the ESAGs found in the generic BES (compare fig. 1a and 1b). Instead T.b. rhodesiense has a serum resistance associated (SRA) ESAG which confers resistance against human immune defenses (VanHamme, et al., 2001).

Metacyclic VSG ESs (MESs) are monocistronic with only a few or degenerate ESAGs. MESs also have fewer repetitive sequences than BESs and therefore are less recombinationally active (Ersfeld, et al., 1999). MESs are activated through in situ activation, which is stimulated by specific factors from the fly (VanHamme & Pays, 1995).

The function of most ESAGs is unknown although a few may be involved in the transformation from the bloodstream to procyclic form. Some ESAGs contain transmembrane domains, GPI anchors, and amino-terminal signal peptides. The following table illustrates the little that is known about structures, protein associations or functions of the products of certain ESAGs according to VanHamme, et al. (2001).

Gene Product from Structure or function
ESAG8 Interacts with PIE8, which is highly concentrated in mitochondrion of bloodstream forms.
ESAG4 Causes increase in adenylyl cyclase activity involved in transformation from the bloodstream slender to stumpy to procyclic forms.
ESAG10 Ten transmembrane domains, possibly involved in pteridin transport.
ESAG6/7 Homologous & products form heterodimers. Tether to membrane with a GPI anchor from carboxyl end, amino terminus binds transferrin.
SRA Confers resistance to trypanosome lytic factor from human serum.

 

VSG Gene Regulation of the Bloodstream Form

Having several BESs increases the trypanosome’s potential to adapt to many changes in the environment by modifying its antigen surface. These modifications are subtle because BESs are 90% homologous (VanHamme, et al., 2001).

Activation of a particular VSG gene primarily depends on its incorporation into a BES by reciprocal recombination. Recombination events are favored because of high sequence homologies between BESs and high levels of repetition within BESs (VanHamme, et al., 2001).

Although all BESs have activated promoters, only one is expressed at any time. Repression of silent BESs is thought to occur through blocking of the elongation phase of transcription through situ activation/inactivation. This may involve chromatin remodeling, subnuclear localization or the distance from the telomere. Chromosomal repression may target the elongation factors associated with RNA processing, nuclear export, and RNA stabilization that must occur at the active site. The presence of the novel base ß-D-glucosylhydroxymethyluracil, otherwise known as base J, may regulate chromosomal repression. Base J is found in repeats flanking active expression sites and is associated with heterochromatin-like chromatin organization (VanHamme, et al., 2001).

 

Differentiation from the Bloodstream to the Procyclic Form

The stumpy forms will die unless they are ingested by the tsetse fly. When stumpy forms are placed under the appropriate conditions to differentiate into the procyclic form VSG expression is terminated. VSG mRNA is preferentially degraded and VSGs are released from the cell surface by protease cleavage at the base of the antigen (VanHamme & Pays, 1995).


Sources:

Ersfeld, K., Melville, S.E, & Gull, K. (1999). Nuclear and genome organization of Trypanosoma brucei. Parasitology Today 15: 58-64.

VanHamme, L. & Pays, E. (1995). Control of gene expression in trypanosomes. Microbiological Reviews 59: 223-240.

Vanhamme L., Lecordier L., & Pays E. (2001). Control and function of the bloodstream variant surface glycoprotein expression sites in Trypanosoma brucei. International Journal of Parasitology, 31, 523-531.

Links:

Trypanosoma brucei genome network

 

Created by Margaret Maddux as part of a senior seminar at Earlham College

Last updated: April 10, 2003