A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Genes & development (Impact Factor: 12.64). 01/2011; 25(2):137-52. DOI: 10.1101/gad.1980311
Source: PubMed

ABSTRACT Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci.

Download full-text


Available from: Julie A Law, Nov 21, 2014
  • Source
    • "Most algal KMT1 proteins show high sequence similarity to land plant KMT1 polypeptides, both within the SET domain and in the surrounding regions known as the Pre-SET and Post-SET motifs (Fig. 1). Additionally, all algal sequences contain an SRA (SET and RING associated) domain (Fig. 1), which recognizes the methylation status of CG and CHH DNA sequences (where H = A, T, or C) (Rajakumara et al., 2011). Land plant KMT1 proteins have been reported to fall into several distinct subgroups, indicative of functional diversification (Casas-Mollano et al., 2007; Huang et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microalgae exhibit enormous diversity and can potentially contribute to the production of biofuels and high value compounds. However, for most species, our knowledge of their physiology, metabolism, and gene regulation is fairly limited. In eukaryotes, gene silencing mechanisms play important roles in both the reversible repression of genes that are required only in certain contexts and the suppression of genome invaders such at transposons. The recent sequencing of several algal genomes is providing insights into the complexity of these mechanisms in microalgae. Collectively, glaucophyte, red, and green microalgae contain the machineries involved in repressive histone H3 lysine methylation, DNA cytosine methylation, and RNA interference. However, individual species often only have subsets of these gene silencing mechanisms. Moreover, current evidence suggests that algal silencing systems function in transposon and transgene repression but their role(s) in gene regulation or other cellular processes remains virtually unexplored, hindering rational genetic engineering efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Bioresource Technology 10/2014; 184. DOI:10.1016/j.biortech.2014.10.119 · 5.04 Impact Factor
  • Source
    • "Additionally, an extra SRA (SET-or RING-associated) methyl binding domain is present at the N termini of SU(VAR)3-9 homologs (Baumbusch et al., 2001). The 5-methyl-cytosine binding ability of the SRA domain is required for both DNA methylation and H3K9me2 (Rajakumara et al., 2011). The cooperation between DNA methylation and histone methylation is essential for maintaining stable chromatin silencing in Arabidopsis (Baubec et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.
    The Plant Cell 07/2013; DOI:10.1105/tpc.113.114678 · 9.58 Impact Factor
  • Source
    • "Zfp57 and MBD proteins share a common mode of 5mC recognition Two other protein domains—namely, SET and RINGassociated (SRA) domain (Arita et al. 2008; Avvakumov et al. 2008; Hashimoto et al. 2008; Rajakumara et al. 2011) and MBD (Ohki et al. 2001; Ho et al. 2008; Scarsdale et al. 2011)—have been structurally characterized in their recognition of 5mC. The mammalian SRA domain flips the 5mC of a hemimethylated CpG into a binding cage with the methyl group in hydrophobic interactions with the side chains of two tyrosines and one serine (Supplemental Fig. S3A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Zinc finger transcription factor Zfp57 recognizes the methylated CpG within the TGCCGC element. We determined the structure of the DNA-binding domain of Zfp57, consisting of two adjacent zinc fingers, in complex with fully methylated DNA at 1.0 Å resolution. The first zinc finger contacts the 5' half (TGC), and the second recognizes the 3' half (CGC) of the recognition sequence. Zfp57 recognizes the two 5-methylcytosines (5mCs) asymmetrically: One involves hydrophobic interactions with Arg178, which also interacts with the neighboring 3' guanine and forms a 5mC-Arg-G interaction, while the other involves a layer of ordered water molecules. Two point mutations in patients with transient neonatal diabetes abolish DNA-binding activity. Zfp57 has reduced binding affinity for unmodified DNA and the oxidative products of 5mC.
    Genes & development 10/2012; 26. DOI:10.1101/gad.202200.112 · 12.64 Impact Factor
Show more