Roles Of Human Elongator Complex And Its HAT Activity In Gene Regulation

The packaging of eukaryotic DNA into chromatin influences various processes that utilize DNA as a template, including transcription, replication and repair. Eukaryotic messenger RNAs are synthesized by the multisubunit enzyme RNA polymerase II, aided by myriad cofactors that control different events in this multistep process. A complete round of transcription involves the recruitment of polymerase and general transcription factors to the promoter, RNA chain synthesis initiation and polymerase escape form the promoter, RNA chain elongation, and finally termination with relaease of polymerase and nascent transcript from the DNA template. As RNA polymerase II leaves a gene promotor to transcribe the coding region, it faces many obstacles, including nucleosomes. Recent data have demonstrated that the tails of histones H3 and H4 greatly reduce the speed of transcript elongation through a nucelosome, and tail acetylation suppresses this effect. Accessory factors for elongating polymerase would be expected to carry out these modifications. The RNAPII -associated complex Elongator has histone acetyltransferase (HAT) activity and thus might fulfil such a role.Yeast Elongator was isolated as a complex that associates with the chromatin fraction and interacts with the elongating phosphorylated form of RNAP II. The functional entity of Elongator complex has recently been shown to be an unstable six-subunit complex, termed holo-Elongator, which can dissociate into two discrete three-subunit subcomplexes upon treatment with high salt and /or MonoQ chromatography. One of these subcompexes is the Elp3-containing core complex, and the other is a complex of the Elp4, Elp5 and Elp6 proteins. Deletion of ELP3 (or other ELP genes) confers a set of phenotypes, some of which are associated with elongation defects. Significantly, the Elp3 subunit is a highly conserved histone acetyltransferase (HAT). Mutations that debilitate the HAT activity of EIp3 in vitro also confer elp phenotypes in vivo, indicating that the HAT activity of Elongator is required for its function. The activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, the predominant acetylation sites are lysine-14 ofhistone H3 and lysine-8 of histone H4. The small subcomlexes composed of Elp4/5/6 is required for the HAT activity, it interacts preferentially with the core complex(rather than in complex with RNAPII) and was therefore proposed to have a regulatory function.Recently, human Elongator complexes have been isolated. The human complex is a fragile six-subunit complex that interacts with RNAP II and has HAT activity directed against histone H3 and H4. Depletion of Elongator from HeLa nuclear extracts reduces the ability of these extracts to transcribe chromatin templates, providing biochemical support for the proposed role of Elongator during transcript elongation in chromatin. However all of these data come from in vitro studies. There have been no reports so far on the characterization of human Elongator complex in vivo.In this thesis, we studied the function of HAT activity of human Elongator complex in gene expression and regulation in Saccharomyces cerevisiae. The results have shown that the HAT activity ofELP3, which is essential for its function in vivo, is evolutionarily conserved between human and yeast. Human Elongator complex serves a role in chromatin remodeling through modifying the lysine residues of histone H3 and H4 during transcription elongation in higher cells. The main results and conclusions are as followings.1 Using depletion strain of yeast as material, we have established a system for complementation test and gene expression analysis of human Elongator complex.2, By using the above system, we demonstrated that yELP3, yhELP3 and hELP3 were able to completely, dramatically and partially complement the growth defects and the slow activation of PHO5 and SSA3 gene caused by the depletion of yELP3, respectively, while yhELPBHAT' whose catalytic domain was partially de...