组蛋白甲基化和乙酰化修饰对基因表达和细胞生长至关重要,为揭示组蛋白H3第4、36位赖氨酸(K)修饰对酵母生长和诱导基因表达的重要性及两位点功能差异,文章构建了两位点单独或共同突变为亮氨酸(L)的组蛋白突变株S4、S36和D436,对其在正常、半乳糖为单一碳源、高温、高盐等条件下的生长及GAL1、SSA3和PHO5表达进行比较。结果显示：D436对高温最敏感,各突变株对咖啡因显著敏感;3个突变株在高温、高盐、6-AU、咖啡因存在时的生长及GAL1、SSA3和PHO5的激活均明显慢于野生型;S4在高温、高盐条件下生长及GAL1激活慢于S36。H3-K4和H3-K36的翻译后修饰对细胞生长和适应不利环境非常重要,在对高温等逆境快速适应上,K4比K36更重要,组蛋白突变株的表型缺陷是因该条件下细胞生存所必需的诱导基因表达延迟所致,同一位点突变对不同基因表达有不同影响。3个突变株的缺陷表型严格上应是相应位点突变导致组蛋白修饰模式改变所造成的综合影响。
Post-translational modifications of histones, such as acetylation and methylation, have a pivotal role in regulating gene expression and cell growth. To elucidate the different roles and importance of H3K4 and H3K36 modifications in expression of inducible genes such as Cal1, SSA3, PHO5 and the growth of yeast cell, we constructed three different yeast mutant strains carrying mutations of lysine 4, 36, or both to leucine in the histone H3 tail. Real-time PCR and sensitive assay under the conditions of high temperature, NaCl, caffeine, 6-AU, or other conditions were carried out to characterize the effects of these mutations on cell growth and transcription levels of GAL1, SSA3 and PHO5. The results showed that three histone methylation mutants exhibited more severe growth defects and slower activation of GAL1, SSA3 and PHO5 than those of wild type; H3K4L/H3K36L double mutant strain D436 has the most severe phenotype. H3K4L mutants S4 exhibited more severe defects than those of H3K36L S36 mutants, especially at high temperature and high NaCl stresses. These results show that H3K4L and H3K36L are important for the growth and survival of yeast in unfavorable conditions, and that different mutations have different effects on the expression of single inducible gene, whereas the same mutation has different effects on the activation of different inducible genes in vivo. The post-translational modification of H3K4 is more important than H3K36 on the adaptation to harsh condition for yeast cell. The growth defects of histone mutant strains might arise from the slow activation of inducible gene essential for survival at harsh conditions.
[1] H, Schneider R, Bannister AJ, Sherriff J, Bernstein BE, Emre NC, Schreiber SL, Mellor J, Kou-zarides T. Active genes are tri-methylated at K4 of histone H3. Nature , 2002, 419(6905): 407-411.
[2] NJ, Kim M, Tong A, Golshani A, Cagney G, Canadien V, Richards DP, Beattie BK, Emili A, Boone C, Shilatifard A, Buratowski S, Greenblatt J. Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol Cell Biol , 2003, 23(12): 4207-4218.
[3] AJ, Schneider R, Myers FA, Thorne AW, Crane-Robinson C, Kouzarides T. Spatial distribution of di- and tri-methyl lysine 36 of histone H3 at active genes. J Biol Chem , 2005, 280(18): 17732-17736.
[4] 黄秋花. 组蛋白赖氨酸甲基化在表观遗传调控中的作用. 遗传, 2007, 29(4): 387-392.
[5] M. Nucleosomes: regulators of transcription. Trends Genet , 1990, 6(12): 395-400.
[6] MM. Histone structure and function. Curr Opin Cell Biol , 1991, 3(3): 429-437.
[7] SY. Chromatin-mediated transcriptional repression in yeast. Curr Opin Genet Dev , 1995, 5(2): 168-173.
[8] J, Frisch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989: 19-20, 55-56, 880-885.
[9] J, Donald KA, Griffiths DE, Donald G. DMSO-en-hanced whole cell yeast transformation. Nucleic Acids Res , 1991, 19(20): 5791.
[10] 田树娟. 双元载体提供组蛋白H3/H4拷贝1的elp3Δ菌株的构建. 河南师范大学学报(自然科学版), 2008, 36(2): 95-98.
[11] F, Lu J, Han QJ, Zhang GP, Huang BQ. The Elp3 subunit of human Elongator complex is functionally similar to its counterpart in yeast. Mol Genet Genomics , 2005, 273(3): 264-272.
[12] 孔艳, 张帅, 马瑜, 胡岩岩. 组蛋白不同乙酰化位点突变对酵母细胞生长和 SSA3 、 GAL1 基因表达的影响. 中国生物化学与分子生物学报, 2010, 26(9): 822-827.
[13] F, Ma JX, Ma Y, Tian SJ, White RE, Han GC. hElp3 Dir-ectly modulates the Expression of HSP70 gene in HeLa cells via HAT activity. PLOS O NE , 2011, 6(12): e29303.
[14] KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods, 2001, 25(4): 402-408.
[15] BD, Allis CD. The language of covalent histone modifications. Nature , 2000, 403(6765): 41-45.
[16] K, Shilatifard A. A COMPASS in the voyage of defining the role of trithorax/MLL-containing complexes: linking leukemogensis to covalent modifications of chromatin. J Cell Biochem , 2005, 95(3): 429-436.
[17] M, Shilatifard A. Transcriptional elongation by RNA polymerase II and histone methylation. J Biol Chem , 2003, 278(29): 26303-26306.
[18] C, Cui GF, Botuyan MV, Mer G. Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S. Structure, 2008, 16(11): 1740-1750.
[19] A, Karabetsou N, Nair A, Mellor J. Dynamic lysine methylation on histone H3 defines the regulatory ph-ase of gene transcription. Molr Cell , 2005, 18(6): 723-734.
[20] MJ, Li B, Florens L, Suganuma T, Swanson SK, Lee KK, Shia WJ, Anderson S, Yates J, Washburn MP, Workman JL. Histone H3 methylation by Set2 directs dea-cetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell , 2005, 123(4): 581-592.
[21] S, Smolle M, Li H, Gogol MM, Saint M, Ku-mar S, Natarajan K, Workman JL. Set2 methylation of his-tone H3 lysine 36 suppresses histone exchange on transcribed genes. Nature, 2012, 489(7416): 452-455.
[22] A, Utley RT, Allard S, Côté J. Recruitment of the NuA4 complex poises the PHO5 promoter for chromatin remodeling and activation. EMBO J , 2004, 23(13): 2597- 2607.
[23] CD, Kladde MP. Effectors of lysine 4 methylation of histone H 3 in Saccharomyces cerevisiae are negative regulators of PHO5 and GAL1-10. J Biol Chem , 2004, 279(32): 33057-33062.
[24] BD, Ohba R, Cook RG, Allis CD. Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci USA , 1999, 96(26): 149
