Both combinatorial K4me0-K36me3 marks on sister histone H3s of a nucleosome are required for Dnmt3a-Dnmt3L mediated de novo DNA methylation

a.
State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China
b.
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China

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Corresponding author: E-mail address: jqzhou@sibcb.ac.cn (Jin-Qiu Zhou)

These authors contribute equally to this work.

摘要

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Abstract: A nucleosome contains two copies of each histone H2A, H2B, H3 and H4. Histone H3 K4me0 and K36me3 are two key chromatin marks forde novo DNA methylation catalyzed by DNA methyltransferases in mammals. However, it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively. Here, taking advantage of the bivalent histone H3 system in yeast, we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L. The results show that lack of both K4me0 and K36me3 on one sister H3 tail, or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC, revealing a synergy of two sister H3s in DNA methylation regulation. Accordingly, the Dnmt3a or Dnmt3L mutation that disrupts the interaction of ^Dnmt3aADD domain-H3K4me0, ^Dnmt3LADD domain-H3K4me0, or ^Dnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation. These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s, and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.
- Asymmetrical nucleosome /
- Histone H3K4 methylation /
- Histone H3K36 methylation /
- Yeast
These authors contribute equally to this work.
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Fig. 1. Expression of murine Dnmt3a and Dnmt3L in H3^D/H3^H yeast strain results in DNA methylation. A: Schematic representation of yeast strain carrying both hht1-A110D (H3^D) and hht1-L110H (H3^H) genes, as well as yeast strain transformed with plasmids bearing genes encoding Dnmt3a and Dnmt3L. B: Western blot analysis to detect the co-expression of HA-tagged Dnmt3a (α-HA) and Flag-tagged Dnmt3L (α-Flag) in four individual clones of H3^D/H3^H strain cultured in glucose (Glu) or galactose (Gal) media. Blotting with antibodies against α-tubulin served as a loading control. C: HPLC analysis of the 5-methyl cytosine (5mC) level in yeast genomic DNA. Representative elution profiles of cytosine (C) and 5mC are shown. D: Quantification of 5mC in H3^D/H3^H strain co-expressing Dnmt3a and Dnmt3L. Error bars represent SD (n = 3).

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Fig. 2. Bisulfate sequencing and nucleosome positioning in H3^D/H3^H strain with overexpression of Dnmt3a and Dnmt3L. A and B: Bisulfate sequencing detection of 5mC at the genomic loci of YNL106C (A) and YPL017C (B). Unmethylated and methylated CpG sites are shown with open and filled circles, respectively. C and D: Nucleosome positioning in YNL106C (C) and YPL017C (D). Upper panel: Mononucleosome DNA samples digested by three titrations of MNase (60 U, 120 U and 200 U) were used as template for qPCR. The numbers of X-axis are corresponding to the YNL106C or YPL017C locus; the relative protection values shown in Y-axis are normalized to the value obtained from CEN3. Lower panel: Amplicons (indicated by arrows) of the YNL106C and YPL017C loci are used for nucleosome scanning assay (NuSA). Amplicons of A12/A13, A17/A17’ and A21/A22 for YNL106C and amplicons of A2/A3, A6/A8 and A11/A12 for YPL017C separately indicate three regions that are relatively less sensitive to MNase digestion (upper panel). The positions of three nucleosomes are schematically indicated by ovals (lower panel). Light blue bar indicates the region (1059 to 1640 of YNL106C and 353 to 911 of YPL017C) examined by bisulfite sequencing. Vertical blue lines indicate CpG sites in the YNL106C and YPL017C loci, and closed circles on top of blue lines indicate methylated CpG sites detected in (A) and (B).

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Fig. 3. Histone H3K4A and H3K36A mutations cause reduction of 5mC level. A and D: Western blot analysis of the expression of HA-Dnmt3a and Flag-Dnmt3L in serial H3K4A (A) and H3K36A (D) mutants upon galactose induction. B and E: Western blot analysis of H3K4me3 levels in serial H3K4A mutants (B) and H3K36me3 levels in serial H3K36A mutants (E). In E, arrowhead indicates the band corresponding to H3K36me3. C and F: 5mC levels in serial H3K4A (C) and H3K36A (F) mutants. Error bars represent SD (n = 3).

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Fig. 4. SET1 deletion promotes while SET2 deletion attenuates DNA methylation. A: Western blot analysis of the expression of HA-Dnmt3a and Flag-Dnmt3L in the set1Δ H3^D/H3^H, set2Δ H3^D/H3^H and set1Δ set2Δ H3^D/H3^H mutant strains. B: Western blot analysis of H3K4me3 and H3K36me3 levels in the set1Δ H3^D/H3^H, set2Δ H3^D/H3^H and set1Δ set2Δ H3^D/H3^H mutant strains. C: 5mC levels in the set1Δ H3^D/H3^H, set2Δ H3^D/H3^H and set1Δ set2Δ H3^D/H3^H mutant strains. Error bars represent SD (n = 3). D: Bisulfate sequencing detection of 5mC in the genomic locus of YNL106C in the set1Δ H3^D/H3^H, set2Δ H3^D/H3^H and set1Δ set2Δ H3^D/H3^H mutant strains. E: 5mC levels in serial H3K36A mutants in set1Δ background. Error bars represent SD (n = 3). F: Bisulfate sequencing detection of 5mC in the genomic loci of YNL112W and YNL104C in serial set1Δ H3K36A mutants. In D and F, unmethylated and methylated CpG sites are shown with open and filled circles, respectively.

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Fig. 5. Combinatorial mutations of H3K4A and H3K36A in one and/or two sister histone H3s greatly reduced 5mC. A: 5mC levels in the isogenic strains labeled at bottom. B: 5mC levels in the isogenic strains labeled at bottom upon SET1 deletion. Error bars represent SD (n = 3).

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Fig. 6. Expression of Dnmt3a^D527A, Dnmt3L^I141W or Dnmt3a^S333P results in significant reduction of 5mC level. A: 5mC levels in the H3^D/H3^H and set1Δ H3^D/H3^H strains expressing Dnmt3a/Dnmt3L, Dnmt3a^D527A/Dnmt3L, Dnmt3a/Dnmt3L^I141W, Dnmt3a^D527A/Dnmt3L^I141W and Dnmt3a^S333P/Dnmt3L. Dnmt3a^D527A, a Dnmt3a ADD domain point mutation; Dnmt3L^I141W, a Dnmt3L ADD domain point mutation; Dnmt3a^S333P, a Dnmt3a PWWP domain point mutation. Error bars represent SD (n = 3). B: Simplified working model of how two Dnmt3a-Dnmt3L heterodimers bind two tails of sister histone H3s in one nucleosome. Each H3 tail possesses both K4me0 and K36me3 marks. The^Dnmt3LADD domain binds the H3K4me0 mark, while the ^Dnmt3aPWWP domain binds the H3K36me3 mark (upper panel). The H3K4me0 mark can be transferred to the ^Dnmt3aADD domain (lower panel). C: Simplified working model of how two Dnmt3a-Dnmt3L heterodimers bind two tails of H3s from two proximal nucleosomes. Each H3 tail possesses both K4me0 and K36me3 marks. The ^Dnmt3LADD domain binds the H3K4me0 mark, while the ^Dnmt3aPWWP domain binds the H3K36me3 mark. The association of the ^Dnmt3aADD domain with H3K4me0 is not depicted.

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参考文献(40)

[1]	Baubec, T., Colombo, D.F., Wirbelauer, C., Schmidt, J., Burger, L., Krebs, A.R., Akalin, A., Schubeler, D., 2015. Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature 520, 243-247.
[2]	Bauer, I., Graessle, S., Loidl, P., Hohenstein, K., Brosch, G., 2010. Novel insights into the functional role of three protein arginine methyltransferases in Aspergillus nidulans. Fungal Genet. Biol. 47, 551-561.
[3]	Bestor, T.H., 2000. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9, 2395-2402.
[4]	Capuano, F., Mulleder, M., Kok, R., Blom, H.J., Ralser, M., 2014. Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species. Anal. Chem. 86, 3697-3702.
[5]	Chedin, F., Lieber, M.R., Hsieh, C.L., 2002. The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc. Natl. Acad. Sci. U. S. A. 99, 16916-16921.
[6]	Chen, T., Tsujimoto, N., Li, E., 2004. The PWWP domain of Dnmt3a and Dnmt3b is required for directing DNA methylation to the major satellite repeats at pericentric heterochromatin. Mol. Cell. Biol. 24, 9048-9058.
[7]	Dhayalan, A., Rajavelu, A., Rathert, P., Tamas, R., Jurkowska, R.Z., Ragozin, S., Jeltsch, A., 2010. The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation. J. Biol. Chem. 285, 26114-26120.
[8]	Edwards, J.R., Yarychkivska, O., Boulard, M., Bestor, T.H., 2017. DNA methylation and DNA methyltransferases. Epigenet. Chromatin 10, 23.
[9]	Ge, Y.Z., Pu, M.T., Gowher, H., Wu, H.P., Ding, J.P., Jeltsch, A., Xu, G.L., 2004. Chromatin targeting of de novo DNA methyltransferases by the PWWP domain. J. Biol. Chem. 279, 25447-25454.
[10]	Gietz, R.D., Schiestl, R.H., 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31-34.
[11]	Gowher, H., Stockdale, C.J., Goyal, R., Ferreira, H., Owen-Hughes, T., Jeltsch, A., 2005. De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases. Biochemistry 44, 9899-9904.
[12]	Guo, X., Wang, L., Li, J., Ding, Z., Xiao, J., Yin, X., He, S., Shi, P., Dong, L., Li, G., Tian, C., Wang, J., Cong, Y., Xu, Y., 2015. Structural insight into autoinhibition and histone H3-induced activation of DNMT3A. Nature 517, 640-644.
[13]	Holz-Schietinger, C., Matje, D.M., Harrison, M.F., Reich, N.O., 2011. Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation. J. Biol. Chem. 286, 41479-41488.
[14]	Hu, J.L., Zhou, B.O., Zhang, R.R., Zhang, K.L., Zhou, J.Q., Xu, G.L., 2009. The N-terminus of histone H3 is required for de novo DNA methylation in chromatin. Proc. Natl. Acad. Sci. U. S. A. 106, 22187-22192.
[15]	Huang, H., Lin, S., Garcia, B.A., Zhao, Y., 2015. Quantitative proteomic analysis of histone modifications. Chem. Rev. 115, 2376-2418.
[16]	Infante, J.J., Law, G.L., Young, E.T., 2012. Analysis of nucleosome positioning using a nucleosome-scanning assay. Methods Mol. Biol. 833, 63-87.
[17]	Jaenisch, R., Bird, A., 2003. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl, 245-254.
[18]	Jia, D., Jurkowska, R.Z., Zhang, X., Jeltsch, A., Cheng, X., 2007. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248-251.
[19]	Jurkowska, R.Z., Jurkowski, T.P., Jeltsch, A., 2011. Structure and function of mammalian DNA methyltransferases. Chembiochem 12, 206-222.
[20]	Kirmizis, A., Santos-Rosa, H., Penkett, C.J., Singer, M.A., Vermeulen, M., Mann, M., Bahler, J., Green, R.D., Kouzarides, T., 2007. Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation. Nature 449, 928-932.
[21]	Law, J.A., Jacobsen, S.E., 2010. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet .11, 204-220.
[22]	Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., Richmond, T.J., 1997. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-260.
[23]	Lund, G., Andersson, L., Lauria, M., Lindholm, M., Fraga, M.F., Villar-Garea, A., Ballestar, E., Esteller, M., Zaina, S., 2004. DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E. J. Biol. Chem. 279, 29147-29154.
[24]	Morselli, M., Pastor, W.A., Montanini, B., Nee, K., Ferrari, R., Fu, K., Bonora, G., Rubbi, L., Clark, A.T., Ottonello, S., Jacobsen, S.E., Pellegrini, M., 2015. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse. eLife 4, e06205.
[25]	Okano, M., Bell, D.W., Haber, D.A., Li, E., 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-257.
[26]	Ooi, S.K., Qiu, C., Bernstein, E., Li, K., Jia, D., Yang, Z., Erdjument-Bromage, H., Tempst, P., Lin, S.P., Allis, C.D., Cheng, X., Bestor, T.H., 2007. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448, 714-717.
[27]	Otani, J., Nankumo, T., Arita, K., Inamoto, S., Ariyoshi, M., Shirakawa, M., 2009. Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain. EMBO Rep. 10, 1235-1241.
[28]	Peng, J., Zhou, J.Q., 2012. The tail-module of yeast Mediator complex is required for telomere heterochromatin maintenance. Nucleic Acids Res. 40, 581-593.
[29]	Rando, O.J., 2010. Genome-wide mapping of nucleosomes in yeast. Methods Enzymol. 470, 105-118.
[30]	Robertson, A.K., Geiman, T.M., Sankpal, U.T., Hager, G.L., Robertson, K.D., 2004. Effects of chromatin structure on the enzymatic and DNA binding functions of DNA methyltransferases DNMT1 and Dnmt3a in vitro. Biochem. Biophys. Res. Commun. 322, 110-118.
[31]	Rondelet, G., Dal Maso, T., Willems, L., Wouters, J., 2016. Structural basis for recognition of histone H3K36me3 nucleosome by human de novo DNA methyltransferases 3A and 3B. J. Struct. Biol. 194, 357-367.
[32]	Shirohzu, H., Kubota, T., Kumazawa, A., Sado, T., Chijiwa, T., Inagaki, K., Suetake, I., Tajima, S., Wakui, K., Miki, Y., Hayashi, M., Fukushima, Y., Sasaki, H., 2002. Three novel DNMT3B mutations in Japanese patients with ICF syndrome. Am. J. Med. Genet. 112, 31-37.
[33]	Smith, Z.D., Meissner, A., 2013. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204-220.
[34]	Suetake, I., Shinozaki, F., Miyagawa, J., Takeshima, H., Tajima, S., 2004. DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction. J. Biol. Chem. 279, 27816-27823.
[35]	Suganuma, T., Workman, J.L., 2008. Crosstalk among histone modifications. Cell 135, 604-607.
[36]	Takeshima, H., Suetake, I., Shimahara, H., Ura, K., Tate, S., Tajima, S., 2006. Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA. J. Biochem. 139, 503-515.
[37]	Takeshima, H., Suetake, I., Tajima, S., 2008. Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1. J. Mol. Biol. 383, 810-821.
[38]	Zhang, Y., Jurkowska, R., Soeroes, S., Rajavelu, A., Dhayalan, A., Bock, I., Rathert, P., Brandt, O., Reinhardt, R., Fischle, W., Jeltsch, A., 2010. Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res. 38, 4246-4253.
[39]	Zhang, Z.M., Lu, R., Wang, P., Yu, Y., Chen, D., Gao, L., Liu, S., Ji, D., Rothbart, S.B., Wang, Y., Wang, G.G., Song, J., 2018. Structural basis for DNMT3A-mediated de novo DNA methylation. Nature 554, 387-391.
[40]	Zhou, Z., Liu, Y.T., Ma, L., Gong, T., Hu, Y.N., Li, H.T., Cai, C., Zhang, L.L., Wei, G., Zhou, J.Q., 2017. Independent manipulation of histone H3 modifications in individual nucleosomes reveals the contributions of sister histones to transcription. eLife 6, e30178.