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

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Abstract
African trypanosomes such asTrypanosoma bruceiundergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing thevariant surface glycoprotein(VSG) gene expressed. The transcribedVSGgene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change theVSGgene 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 thatVSGgenes 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 β-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 telomericVSGexpression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomericVSGgenes in silenced expression sites and not in actively transcribed telomericVSGgenes. J was absent from inactive chromosome-internalVSGgenes. 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 theVSGgene, 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.