Expression-site-associated-gene-8 (ESAG8) is not required for regulation of the VSG expression site in Trypanosoma brucei.

The expression sites (ES) of Trypanosoma brucei are multi-allelic loci from which the Variant Surface Glycoprotein genes (VSG) are expressed. Tight control of the ES transcriptional state ensures that only a single VSG is expressed at a time [1]. Although transcription initiates from multiple ES promoters, only a single ES is completely transcribed [2]. No reproducible differences in DNA sequence [3–5], in chromatin structure [6,7], or in DNA modification of repetitive flanking sequences [8,9] have been detected between the active and silent ESs. Since it is impossible to select for the complete simultaneous transcription of two ESs [10], and since deletion of the active ES promoter precipitates a switch to a previously inactive ES [7], some form of cross-talk is probably responsible for the exclusive transcriptional elongation of the active ES. Current models of ES control postulate the existence of a specific nuclear compartment from which only a single ES can be transcribed, or the presence of a limiting factor that is essential for the productive transcription of the active ES. Co-transcribed from the single ES promoter are multiple expression-site-associated-genes (ESAGs), which provide the parasite with proteins important for growth in the bloodstream (such as the transferrin receptor encoded by ESAGs 6 and 7) and proteins that may function in ES regulation. We have investigated the role of the putative regulatory protein ESAG8 [11,12]. Since different variants of ESAG8 are very highly conserved and appear to be exclusively transcribed from ESs, and because ESAG8 is present exclusively in the bloodstream form at low levels (2000–4000 molecules per cell), we hypothesized that ESAG8 might be involved in ES regulation [13]. ESAG8 consists of an N-terminal RING Zn-finger domain and a C-terminal Leucine Rich Repeat (LRR) domain. The RING Zn-finger motif can mediate the polyubiquitylation and targeted degradation of proteins in several eukaryotic systems [14] and LRR domains are involved in a variety of protein–protein interactions [15]. ESAG8 elutes from a Sephacryl-300 gel filtration column with an apparent molecular weight of approximately 300 kDa, consistent with its presence in a multi-protein complex (Hoek and Cross, submitted for publication). Our recent finding that a small portion of ESAG8 was necessary and sufficient to target the protein to the nucleolus lent further support to the idea that ESAG8 might be involved in ES control, because the ES is transcribed by RNA polymerase I [13,16–18]. To test this hypothesis, we attempted to delete ESAG8 from the actively transcribed ES. In previous experiments, we were unable to target the active ES-linked copy using the highly conserved ESAG8 coding sequence. Targeting was only successful in active ESs containing multiple copies of ESAG8, implying that the gene was essential [13]. We, therefore, designed a conditional disruption strategy to disrupt Abbre iations: ES, expression site; ESAG, expression-site-associated gene; VSG, variant surface glycoprotein. * Corresponding author. Tel.: +1-212-3277-571; fax: +1-2123277-845. E-mail address: george.cross@rockefeller.edu (G.A.M. Cross). 1 Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.