Dissertation > Biological Sciences > Molecular Biology > Molecular Genetics

Effect of Single and Double Mutations of Yeast H3K79L、H4K20L on the Function of ELP3 and Expression of SSA3

Author KongYan
Tutor LiFen
School Henan Normal
Course Cell Biology
Keywords Epigenetics modification Mutation of histone Elongator complex Elp3 Complement test Gene expression
Type Master's thesis
Year 2011
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AIMNESS:Histone modification as one of the key epigenetic regulation mechanisms plays critical roles in various biological processes, including regulation of chromatin structure dynamics and gene expression. Epigenetic modifications, particularly methylation and acetylation or other covalent histone modification, play an important role in regulating chromatin dynamics and therefore have a significant impact on gene expression. It has been showed that the yeast Elongator complex composed of six subunits, as a complex possessing an activity of histone acetyltransferase, facilitates hyperphosphorylated RNAP II to involve in the process of SSA3 and GAL1 transcription elongation. In vitro, the removal of Elongator complex from the HeLa nuclear extracts can decrease the rate of transcription of chromatin templates, which also provides biochemical evidence that human Elongator complex takes part in the transcription elongation. Recently, human Elongator complex has been isolated from HeLa cells and shown to be remarkably similar to its yeast counterpart in either components or functions, The C terminal HAT activity of the catalytic subunit Elp3 is essential to its function in vivo. Bioinformatics analysis showed that the N-terminal of Elp3 has a similar S-adenosylmethionine kinase domain. This domain contains the Fe4S4 cluster and binds SAM, suggesting that it may be related to methylation and thus speculated that the catalytic domain of unknown function may have histone demethylase activity. But the results of Greenwood failed to support a role for Elongator in histone demethylation and support the idea that the Elp3 FeS cluster is essential for normal Elongator function in vivo primarily as a structural, rather than catalytic. Therefore, the second domain of Elp3 function is unclear.Previous studies have indicated that Epigenetic modifications, such as histone acetylated and methylated modifications, have great effects on gene expression and cell growth. Different specific histone lysine methylation (H3K79, H4K20) sites mutation strains were constructed and analysed by yeast complementary experiment and the gene expression detection to investigate the effects of the genome level methylation changes on the fuction of human ELP3, which forms the basis for the further study foucus on the function of the Elongator complex subunit Elp3 and its second catalytic domain activity in the eukaryote gene expression regulation. METHODS:Firstly, the centromere vectors containing designed mutation on specific histone lysine methylation amino sites, such as H3K79, H4K20, were acquired by the site-mutagenesis techinque, and then three mutated elp3Δstrains bearing different methylation sites mutation in H3/H4 were obtained through Plasmid Shuffle assay to replace the wild type histone by desigened mutant histones; and a series of Elp3 plasmids were transformed into the histone mutant elp3Δstrains by LiAc method; Then, complement test at high salt or other different conditions and the SSA3 and GAL1 gene expression analysis were carried out to investigate the effects of single and double mutations of Yeast H3K79L、H4K20L on the function of Elp3 and Expression of SSA3 or GAL1.RESULTS:The main results are as followings.1. The centromere vectors containing specific mutated methylation amino sites such as H3K79, H4K20, were acquired by the site-mutagenesis technique.2. Through Plasmid Shuffle experiments elp3 deletion strains carrying single or double mutations at H3K79, H4K20 and H3K79/H4K20 in histone were constructed.3. Yeast and human Elp3 were transformed into histone mutant elp3Δstrains and complement test and gene expression analysis were carried out. The results showed that mutating the histone methylation amino sites severely reduced the complement ability of yeast and humant Elp3 to the slow growth phenotype and delayed expression of SSA3 and GAL1;4. ELP3 deletion strains carrying sigle mutations on H3K79 had more severe defect phenotypes than ELP3 deletion strains carrying sigle mutation on H4K20, but ELP3 deletion strains carrying sigle mutation on H4K20 had more severe phenotypes than ELP3 deletion strains carrying double mutation on H3K79/H4K20.CONCLUSIONS:The conclusions are as followings.1. The compensation function of yELP3 to the growth defect of elp3Δstrains carrying mutations of lysine-79 in H3, lysine-20 in H4 or concomitant mutation of the two sites into leucine and the slow activation of GAL1 was reduced obviously, and the compensation function of hELP3 was greatly affected, but elp3Δstrains carrying mutations of H3K79/H4K20 did not show more severe phenotypes than elp3Δstrain carrying wild type histone, which indicates that Elp3 may have no direct relationship with the methylation states of H3K20/H4K20.2. The elp3Δstrains carrying single concomitant mutations of H3K79L had more severe phenotypes than ELP3 deletion strains carrying mutation on H4K20L, H4K20L mutant Elp3 deletion srain grows slower than H3K79L/H4K20L concomitant mutant Elp3 deletion strain. These result indicated that different methylation sites have different effect on gene expression and cell growth, maintenance of normal histone H3K79/H4K20 methylation levels in chromatin in vivo is extremely important for the normal growth of cells and also reveals that the effect of yElp3 on the methylation states of H3K79 probably is more important than the methylation states of H4K20 to the normal growth especially to the quickly adapting in altered environments of yeast cells.3. The yELP3 and hELP3 with the deletion of second catalytic domain can not compensate the growth defect on the the elp3Δstrains with histone mutants (H3K79L, H4K20L), and also could not retrieve the delay gene expression of SSA3 and GAL1; it’s showed that the second catalytic domain is crucial to the function of Elp3.

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