A Nucleosome Bridging Mechanism for Activation of a Maintenance DNA Methyltransferase

doi:10.1016/j.molcel.2018.10.006

. 2019 Jan 3;73(1):73-83.e6.

doi: 10.1016/j.molcel.2018.10.006. Epub 2018 Nov 8.

A Nucleosome Bridging Mechanism for Activation of a Maintenance DNA Methyltransferase

Affiliations

¹ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
² Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edyth Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
⁴ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Basic Forestry and Proteomics Research Center, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China; Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Laboratory of Genetics & Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁷ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
⁹ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edyth Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: jacobsen@ucla.edu.
¹⁰ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: geeta.narlikar@ucsf.edu.

PMID: 30415948
PMCID: PMC6407616
DOI: 10.1016/j.molcel.2018.10.006

A Nucleosome Bridging Mechanism for Activation of a Maintenance DNA Methyltransferase

Caitlin I Stoddard et al. Mol Cell. 2019.

. 2019 Jan 3;73(1):73-83.e6.

doi: 10.1016/j.molcel.2018.10.006. Epub 2018 Nov 8.

Authors

Affiliations

¹ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA 94158, USA.
² Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edyth Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
⁴ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Basic Forestry and Proteomics Research Center, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China; Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Laboratory of Genetics & Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁷ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
⁹ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edyth Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: jacobsen@ucla.edu.
¹⁰ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: geeta.narlikar@ucsf.edu.

PMID: 30415948
PMCID: PMC6407616
DOI: 10.1016/j.molcel.2018.10.006

Abstract

DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.

Keywords: DNA methylation; H3K9me; chromatin; heterochromatin; maize; nucleosome; plants.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

**Figure 1.. ZMET2 methylates H3Kc9me dinucleosomes faster than mononucleosomes**
(A) Schematic of full-length ZMET2 domain architecture and crystal structure of truncated ZMET2 (130–912) with bound SAH (PDB 4FSX). Chromodomain is in blue with aromatic cage residue F441 in red, bromo adjacent homology (BAH) domain in green with aromatic cage residue W224 in purple. (B) Activity of ZMET2 for different nucleosomal substrates shown as schematics above bars. All reactions were carried out under single-turnover conditions in which [ZMET2] was in excess and saturating over nucleosomes. Experiments with 601 dinucleosomes (20 bp) were performed in quadruplicate (n=4). Experiments with 601 mononucleosome and 5S dinucleosome were performed in duplicate (n=2). (C) Dinucleosome linker length dependence for ZMET2 activity under single-turnover conditions ([ZMET2] was in excess and saturating over dinucleosomes). For ease of comparison, kcat measurements for dinucleosomes with the 20 bp linker are re-plotted from (A) next to the experiments with dinucleosomes containing 10 bp and 30 bp linkers (n=3) and 40 bp linkers (n=2). (D) DNA methyltransferase activity for WT ZMET2 on ligated nucleosomes with symmetric (two red circles in schematic) or asymmetric (one red, one white circle in schematic) H3Kc9me3 marks (n=6). Reactions were conducted under the conditions as in (C). (E) Activity of ZMET2 for 601 dinucleosomes with 20 bp linkers under multiple-turnover conditions in which the concentration of H3Kc9me3 dinucleosomes is in excess and saturating over the concentration of ZMET2. (F) Dependence of ZMET2 activity on concentration of 601 dinucleosomes with 20 bp linkers. The K_m for H3Kc9me3 dinucleosomes is 340 nM. The K_m for WT dinucleosomes is not reliably measurable due to the low activity (n=2 for all experiments).

**Figure 2.. ZMET2 binding and activity are increased by the H3K9me mark and DNA hemimethylation**
(A) Activity of ZMET2 on 157 bp naked DNA with the 601 sequence in the absence and presence of H3 tail peptides. All peptides contain residues 1–32 of histone H3 and were used at 25 μM. Reactions were conducted under single-turnover conditions in which ZMET2 (5 μM) was in excess and saturating over DNA substrates (200 nM). (B) Rate constants calculated from (A) by dividing initial DNA methylation rates by the concentration of substrate. The values of k_max for no peptide, H3K9me0 and H3K9me2 reactions were 0.00012 (±8.8E-6) min⁻¹, 0.00014 (±1.4E-5) min⁻¹, and 0.011 (±0.0002) min⁻¹, respectively, (n=2 for each measurement). (C) DNA methyltransferase activity of ZMET2 on unmethylated or hemimethylated 38 bp duplexed DNA in the absence (left panel) or presence (right panel) of H3K9me2 peptide. Reactions were conducted as in (D), except with 300 nM 38 bp duplexes. Rate constants for unmethylated, hemimethylated, unmethylated with H3K9me2 and hemimethylated with H3K9me2 were 5.3E-5 (±1.1E-5) min⁻¹, 0.00015 (±3.6E-5) min⁻¹, 0.00049 (±0.00019) min⁻¹, and 0.038 (±0.0095) min ⁻¹, respectively, (n=2 for each measurement). (D) Affinity of ZMET2 for 38 bp duplexed DNA with and without hemimethylation and either H3K9me0 or H3K9me2 peptides measured by fluorescence polarization. Experiments with H3K9me0 peptide were done in duplicate (n=2), unme + H3K9me2 was done in triplicate (n=3), and hemi + H3K9me2 was done in quadruplicate (n=4).

**Figure 3.. The ZMET2 CD recognizes H3K9me in the binding step and the BAH domain recognizes H3K9me in the catalytic step**
(A) WT, F441A and W224L ZMET2 affinity for fluorescein-labeled H3 tail peptides measured by fluorescence polarization. (For these experiments, n=2). (B) WT, F441A and W224L ZMET2 affinity for WT and H3Kc9me3 mononucleosomes measured by fluorescence polarization. Experiments with WT and F441A (CDx) ZMET2 were performed in duplicate (n=2). Experiments with W224L (BAHx) were performed in quadruplicate (n=4). (C) DNA methyltransferase activity for WT, F441A and W224L ZMET2 on WT and H3Kc9me3 dinucleosomes. Reactions were conducted under single-turnover conditions in which the concentration of each ZMET2 protein was in excess and saturating over the dinucleosome concentration. (For these experiments, n=2).

**Figure 4.. ZMET2 preferentially methylates the linker DNA in H3Kc9me3 dinucleosomes**
(A) Schematic of 601 dinucleosome with CHG sites that are either methylated (purple) or not methylated (blue) on the dinucleosome. Only non-CCG sites are colored. Histone H3 is in dark blue (PDB 1ZBB). (B) Schematic of 601 dinucleosome, used to represent approximate structure of a dinucleosome assembled with the 5S positioning sequence. CHG sites that are either methylated (red) or not methylated (green) are labeled. Only non-CCG sites are colored. Histone H3 is in dark blue (PDB 1ZBB). (C) Upper left, bisulfite sequencing time course for ZMET2 activity at 601 CTG 159, the CHG site in the linker region of the dinucleosome, a site where H3Kc9me3 dinucleosomes are methylated faster than naked DNA plus H3K9me2 peptide; upper right, bisulfite sequencing time course for ZMET2 activity at 601 CAG 297, a site where naked DNA plus H3K9me2 peptide is methylated much faster than H3Kc9me3 dinucleosomes; lower left, bisulfite sequencing time course for ZMET2 activity at 601 CAG 209, a site resembling the pattern seen at CAG 297; lower right, bisulfite sequencing time course for ZMET2 activity at 601 CCG 11. This site is representative of all CCG sites in the DNA sequence, in which no DNA methyltransferase activity was detectable. (D) Upper left panel, bisulfite sequencing time course for ZMET2 activity at 5S CAG 161, the CHG site in the linker region of the dinucleosome; upper right panel, bisulfite sequencing time course for ZMET2 activity at 5S CAG 13, a CHG site located near the entry/exit site of the nucleosome; lower left panel, bisulfite sequencing time course for ZMET2 activity at 5S CAG 179, a CHG site located near the entry/exit site of the nucleosome; lower right panel, bisulfite sequencing time course for ZMET2 activity at 5S CAG 110, a site in which methyltransferase activity was much faster on naked DNA plus H3K9me2 peptide than on the dinucleosome. (E) The observed rate constant (k_obs) for DNA methylation on 601 dinucleosomes as measured by the radioactive assay (Figure 1B) is mostly dominated by the rate constant for methylating CHG 159 (k₁₅₉) as measured using bisulfite sequencing. (F) The observed rate constant (k_obs) for DNA methylation on 5S dinucleosomes as measured by the radioactive assay can be explained by contributions from the rate constants of the three most methylated sites as measured by bisulfite sequencing (k₁₆₁, k₁₃, and k₁₇₉).

**Figure 5.. Visualization of ZMET2 bridging the dinucleosome by negative stain electron microscopy**
(A) Two-dimensional class averages of GraFix-treated H3Kc9me3 dinucleosomes (2N_20) alone. The dinucleosomes contain a 20 bp linker with one CHG site positioned 11 bp from one nucleosome and 8 bp from the other. (B) Two-dimensional class averages of GraFix-treated complex formed between H3Kc9me3 dinucleosomes (2N_20) and ZMET2. (C) Two-dimensional class averages depicting different views of the ZMET2/H3Kc9me3 dinucleosome complex. Scale bar is 10 nm. (D) Four different 90° rotational views of a 3D reconstruction of the ZMET2/H3Kc9me3 complex. The complex is represented at two different threshold levels. (E) The same views from (B), with the dinucleosome and ZMET2 (130–912) crystal structures manually fitted into the map. Red arrowheads represent location of emergence of the H3 tail from the globular portion of histone H3 (blue). Colors highlight different domains of ZMET2 (130–912): magenta, catalytic domain; blue, CD; orange, BAH.

**Figure 6.. Model for ZMET2 assembly and activity on chromatin**
(A) ZMET2 interrogates chromatin context by scanning for the correct architecture, and the presence of H3K9me and DNA hemimethylation. In heterochromatic regions of the genome, the ZMET2 CD recognizes the H3K9me mark in the ground state. The ZMET2 BAH domain recognizes the H3K9me mark post-binding in the context of a high-energy intermediate, activating the enzyme for DNA methylation. SAM is utilized for methyl transfer during product formation. (B) Free energy profile for the model depicted in (A). The crosshatch represents the transition state for the chemical step of the reaction. The dotted line represents a lower energy transition state in the presence of a hemimethylated DNA substrate.

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References

1. Anderson JD, Thåström A, and Widom J (2002). Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation. Molecular and Cellular Biology 22, 7147–7157. - PMC - PubMed
1. Bartee L, Malagnac F, and Bender J (2001). Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes & Development 15, 1753–1758. - PMC - PubMed
1. Bernatavichute YV, Zhang X, Cokus S, Pellegrini M, and Jacobsen SE (2008). Genome-Wide Association of Histone H3 Lysine Nine Methylation with CHG DNA Methylation in Arabidopsis thaliana. PLoS ONE 3, e3156–11. - PMC - PubMed
1. Chodavarapu RK, Feng S, Bernatavichute YV, Chen P-Y, Stroud H, Yu Y, Hetzel JA, Kuo F, Kim J, Cokus SJ, et al. (2010). Relationship between nucleosome positioning and DNA methylation. Nature 466, 388–392. - PMC - PubMed
1. Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, and Jacobsen SE (2008a). Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219. - PMC - PubMed

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[1] Anderson JD, Thåström A, and Widom J (2002). Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation. Molecular and Cellular Biology 22, 7147–7157. - PMC - PubMed

[2] Anderson JD, Thåström A, and Widom J (2002). Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation. Molecular and Cellular Biology 22, 7147–7157. - PMC - PubMed

[3] Bartee L, Malagnac F, and Bender J (2001). Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes & Development 15, 1753–1758. - PMC - PubMed

[4] Bartee L, Malagnac F, and Bender J (2001). Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes & Development 15, 1753–1758. - PMC - PubMed

[5] Bernatavichute YV, Zhang X, Cokus S, Pellegrini M, and Jacobsen SE (2008). Genome-Wide Association of Histone H3 Lysine Nine Methylation with CHG DNA Methylation in Arabidopsis thaliana. PLoS ONE 3, e3156–11. - PMC - PubMed

[6] Bernatavichute YV, Zhang X, Cokus S, Pellegrini M, and Jacobsen SE (2008). Genome-Wide Association of Histone H3 Lysine Nine Methylation with CHG DNA Methylation in Arabidopsis thaliana. PLoS ONE 3, e3156–11. - PMC - PubMed

[7] Chodavarapu RK, Feng S, Bernatavichute YV, Chen P-Y, Stroud H, Yu Y, Hetzel JA, Kuo F, Kim J, Cokus SJ, et al. (2010). Relationship between nucleosome positioning and DNA methylation. Nature 466, 388–392. - PMC - PubMed

[8] Chodavarapu RK, Feng S, Bernatavichute YV, Chen P-Y, Stroud H, Yu Y, Hetzel JA, Kuo F, Kim J, Cokus SJ, et al. (2010). Relationship between nucleosome positioning and DNA methylation. Nature 466, 388–392. - PMC - PubMed

[9] Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, and Jacobsen SE (2008a). Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219. - PMC - PubMed

[10] Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, and Jacobsen SE (2008a). Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219. - PMC - PubMed

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A Nucleosome Bridging Mechanism for Activation of a Maintenance DNA Methyltransferase

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A Nucleosome Bridging Mechanism for Activation of a Maintenance DNA Methyltransferase

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