SUMMARY Trypanosoma brucei expresses Variant Surface Glycoprotein (VSG) genes in a strictly monoallelic fashion in its mammalian hosts, but it is unclear how this important virulence mechanism is enforced. Telomere position effect (TPE), an epigenetic phenomenon, has been proposed to play a critical role in VSG regulation, yet no telomeric protein has been identified whose disruption led to VSG derepression. We now identify tbRAP1 as an intrinsic component of the T. brucei telomere complex and a major regulator for silencing VSG expression sites (ESs). Knockdown of tbRAP1 led to derepression of all VSGs in silent ESs, but not VSGs located elsewhere, and resulted in stronger derepression of genes located within 10 kb from telomeres than genes located further upstream. This graduated silencing pattern suggests that telomere integrity plays a key role in tbRAP1-dependent silencing and VSG regulation.

Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interferencemediated knockdown of tbTRF arrested bloodstream cells at G 2 /M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNAbinding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.Telomeres are specialized protein-DNA complexes at the ends of eukaryotic chromosomes. Telomere DNA generally consists of simple, repetitive, TG-rich sequences that are maintained by telomerase (5) and end with a single-stranded G-rich overhang (78). A specialized telomere structure, the T loop, formed by the invasion of telomere G overhangs into the telomeric double-stranded region, in mammals, hypotrichous ciliates, and Trypanosoma brucei has been identified (26,51,53). It is hypothesized that hiding the telomere single-stranded G overhangs in a T-loop structure helps to protect the telomere ends.Proteins that bind to duplex or single-stranded telomere DNA are integral components of the telomere complex and play critical roles in both telomere length regulation and end protection (36). In mammalian cells, two paralogues, TT AGGG repeat-binding factor 1 (TRF1) and TRF2, bind duplex TTAGGG repeats (4,9,12). Both TRF1 and TRF2 have a C-terminal myb motif for DNA binding (3, 9) and an upstream TRF homology (TRFH) domain for homodimerization (24). However, the N termini are quite different: TRF1 is acidic, while TRF2 is basic. Mammalian TRF1 negatively regulates telomere length through a telomerase-dependent pathway (61, 75), whereas TRF2 is involved in both telomere length regulation (38, 61) and telomere end protection (17). Removal of TRF2 from telomeres by overexpression of a dominantnegative mutant of TRF2 resulted in an at least 30%

Very little is known about sequences that mediate nuclear DNA replication in Trypanosoma brucei. We have previously reported the construction and characterization of a panel of autonomously replicating plasmids and artificial linear mini-chromosomes for this organism [1,2]. The episome was obtained by ligating random DNA fragments from the T. brucei genome on to a plasmid that was otherwise incapable of stable replication in this organism. We call these inserted genomic fragments plasmid maintenance sequences (PMS). One member of this panel of episomes (pT13-11) and the corresponding artificial linear mini-chromosomes have been extensively characterized [1][2][3][4]. This plasmid, and by implication its many derivatives (such as pEV-luc; please see below), as well as the linear mini-chromosomes exist at a copy number of 1-2 per cell in procyclic T. brucei and demonstrate substantial mitotic stability in the absence of selection. We are using these autonomous replicons as model systems to delineate critical nuclear DNA replication control elements for this organism.The plasmid pEV-luc ( Figure 1) has been described previously [4]. In this episome, 433 base pairs (-408 to + 25 relative to the start site of transcription) of the GPEET procyclin promoter (also called PARP-A) drives the RNA polymerase I-mediated expression of firefly luciferase and neomycin phosphotransferase (G418 R ). We have previously demonstrated that the autonomous replication of pEV-luc is dependent on the PMS (as expected) and, surprisingly, on the segment bearing the GPEET-promoter [4]. The close association between elements controlling transcription and DNA replication and the possible interaction between factors assembling at these sites is intriguing and fuels our long-term interest in this area. We have sought to delineate the critical promoter-proximal replication control element(s) and determine the overlap (if any) between the two sets of regulatory sequences.The GPEET procyclin promoter has been extensively studied [5][6][7]. It comprises of 4 domains which map to positions -7 to -40 (Domain I), -57 to -72 (Domain II), -90 to -143 (Domain III) and -207 to -222 (Domain IV) with respect to the start point of transcription [5,7; also see Fig 1]. Ten base pair (bp) linker-substitutions within Domains I, II, III and IV reduce GPEETpromotermediated expression of a reporter gene to approximately <0.1%, 5-10%, 30-50%, and 60-70% of wild-type levels respectively [5,7]. Two other RNA Pol I promoters (RRNA and VSG) have been characterized in T. brucei but demonstrate little sequence conservation [8][9][10][11]. Nevertheless, transcription mediated by the GPEET, RRNA or VSG promoters in * Contributed equally to this study ** Corresponding author Tel: 973 720 3454 Fax: 973 720 2338 Email: patnaikp@wpunj.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typeset...