Janzen Christian J, Lander Fabian, Dreesen Oliver, Cross George A M
Laboratory of Molecular Parasitology, The Rockefeller University, Box 185, 1230 York Avenue, New York, NY 10021-6399, USA.
Nucleic Acids Res. 2004 Dec 15;32(22):6575-84. doi: 10.1093/nar/gkh991. Print 2004.
KU is a heterodimer, consisting of approximately 70 and approximately 80 kDa subunits (KU70 and KU80, respectively), which is involved in a variety of nuclear functions. We generated tbKU80-deficient trypanosomes to explore the potential role of the tbKU complex in telomere maintenance and transcriptional regulation of variant surface glycoprotein (VSG) genes in Trypanosoma brucei. Using real-time PCR, we demonstrated that the expression of several different VSG genes remains tightly regulated in tbKU80-deficient bloodstream-form cell lines, suggesting that VSG transcription profiles do not change in these cells. Owing to developmental silencing of the VSG Expression Sites (ES), no VSG is transcribed in the insect procyclic stage. With a green fluorescent protein reporter system, we showed that tbKU80-deficient mutants are fully capable of ES silencing after differentiation into procyclic forms. Using T7 RNA polymerase to explore the transcriptional accessibility of ES chromatin in vivo, we demonstrated that tbKU80-deficient bloodstream-form cells were able to generate transcriptionally repressed ES chromatin after differentiation into procyclic cells. Finally, we demonstrated progressive telomere shortening in tbKU80-deficient mutants. The possible function of tbKU80 in telomere maintenance and regulation of telomerase is discussed.
KU是一种异源二聚体，由大约70 kDa和大约80 kDa的亚基（分别为KU70和KU80）组成，参与多种核功能。我们构建了tbKU80缺陷型锥虫，以探索tbKU复合物在布氏锥虫端粒维持和可变表面糖蛋白（VSG）基因转录调控中的潜在作用。通过实时PCR，我们证明在tbKU80缺陷型血流形式细胞系中，几种不同VSG基因的表达仍受到严格调控，这表明这些细胞中的VSG转录谱没有变化。由于VSG表达位点（ES）的发育沉默，在昆虫前循环阶段没有VSG被转录。利用绿色荧光蛋白报告系统，我们表明tbKU80缺陷型突变体在分化为前循环形式后完全能够实现ES沉默。利用T7 RNA聚合酶在体内探索ES染色质的转录可及性，我们证明tbKU80缺陷型血流形式细胞在分化为前循环细胞后能够产生转录抑制的ES染色质。最后，我们证明了tbKU80缺陷型突变体中端粒的逐渐缩短。讨论了tbKU80在端粒维持和端粒酶调控中的可能功能。
