Regulation of Trypanosome DNA Glycosylation by a SWI2/SNF2-like Protein

Abstract

Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

“…The in vivo analysis of JGT function in T. brucei further supports this hypothesis. We have demonstrated previously that both JBP1 and JBP2 can stimulate de novo synthesis of hmU, which is subsequently converted to base J (9,14,24). Upon loss of JGT activity, we would expect de novo activities of JBP1/2 to modify the same sites as in WT cells but without having the hmU converted to J. Trypanosomatids lack DNA glycosylases (i.e.…”

“…Trypanosome Cell Culture and Generation of T. brucei Transfectants-The bloodstream form T. brucei cell line strain 427 and the 90 -13 RNAi cell line were cultured in HMI9 medium supplemented with 10% heat-inactivated fetal bovine serum and 10% serum plus as described previously (9). 90-13 cells were cultured in the presence of 2.5 g/ml neomycin and 5 g/ml hygromycin to maintain the intergrated genes for T7 RNA polymerase and the tetracycline repressor, respectively.…”

“…JBP2 does not bind the modified base directly, but is able to bind chromatin in a base J-independent manner, presumably via the C-terminal SWI2/SNF2 domain (9). Although both JBP1 and JBP2 stimulate de novo thymidine hydroxylation in vivo, the ability of JBP1 to bind J-DNA is thought to play a role in J propagation/maintenance (9,14,19). Deletion of either JBP1 or JBP2 from the bloodstream form T. brucei results in a 20-and 8-fold reduction in the levels of base J, respectively (10,14,20).…”

JBP1 and JBP2 Proteins Are Fe2+/2-Oxoglutarate-dependent Dioxygenases Regulating Hydroxylation of Thymidine Residues in Trypanosome DNA

“…In fact, the low level of in vitro conversion of thymidine residues suggests that the ability of JBP1 to bind J residues in dsDNA may enhance TH activity. This "propagation" activity may represent an essential function for JBP1 in vivo (7,35). We have previously demonstrated that JBP1 and JBP2 operate optimally in different chromatin environments; JBP1 at internal regions involved in Pol II transcription and JBP2 within telomeric-localized repetitive DNA (2).…”

“…Cell Culture-Blood stream form T. brucei cell line 221a of strain 427 were cultured as described previously (7). Promastigote L. major cells were grown at 26°C in M199 media supplemented with 10% FBS as described (19).…”