Localization of the modified base J in telomericVSGgene expression sites ofTrypanosoma brucei

Abstract

African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base ␤-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.

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.…”

“…Determination of the Genomic Level of J-To quantify the genomic J levels, DNA was isolated and utilized in the anti-J DNA immunoblot assay as described previously (24). Briefly, serially diluted genomic DNA was blotted to nitrocellulose, followed by incubation with anti-J antisera.…”

FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

“…43,44 Moreover, base J is suggested to play a role in gene silencing related to the process of antigenic variation. 45 Indirect evidence suggests that base J is synthesized in two steps (Scheme 7). 46,47 First, the methyl group of thymidine must be hydroxylated to create hydroxymethyluridine (HOMedU), as evidenced by the presence of low levels of HOMedU in DNA of bloodstream form trypanosomes.…”

Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

“…Analysis of these marked expression sites after in situ switches have not shown detectable obligatory DNA rearrangements , Horn & Cross, 1997a, Rudenko et al 1998a). In addition, the unique marker genes inserted have been used to probe the expression site promoter area for J, a unique DNA modification found in trypanosomatids (Van Leeuwen et al 1997).…”