The SRA methyl-cytosine-binding domain links DNA and histone methylation - PubMed (original) (raw)

Comparative Study

Lianna M Johnson et al. Curr Biol. 2007.

Abstract

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.

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Figures

Figure 1

Figure 1

Correlation of DNA and histone methylation in Arabidopsis. (A) Bisulfite sequencing data for wild type, drm1 drm2 cmt3 and met1 at AtSN1, expressed as the average number of methylated CG, CNG or CNN residues per clone. (B) ChIP analysis using antibodies specific for H3K9me2 from the wild type, drm1 drm2 cmt3, met1 and kyp-6. AtSN1 was amplified from the IP and no antibody control (no AB). ACTIN was amplified from the IP. The relative quantity precipitated for each sample was derived from a standard curve of input DNA. The average of two biological replicas done in duplicate is shown with standard deviation. (C) Bisulfite sequencing data from AtCOPIA4. The remaining non-CG methylation in the drm1 drm2 cmt3 triple mutant is discussed in reference [19]. (D) ChIP analysis of H3K9me2 at AtCOPIA4. The average of two biological replicas done in duplicate is shown with standard deviation.

Figure 2

Figure 2

The SRA domain. (A) ClustalW alignment of the SRA domains from the nine SUVH genes, two Arabidopsis SRA- and RING-domain-containing proteins, mouse np95, and human ICBP90. The site of the KYP S200F[4] mutation is indicated with “#”, the E208K mutation is indicated with a “*”, and the R260H[4] mutation is indicated with a “+”. Arrows show the SRA domain identified by SMART and the boxed in amino acids are those included in the GST-SUVH6-SRA fusion protein. The association of SRA domains with SET domain proteins is unique to the plant kingdom. (B) Immunofluorescence of H3K9me2 compared to DAPI in nuclei isolated from wild type, kyp-2 (null allele), and kyp-5 (SRA mutation E208K). (C) Regions of the SUVH genes contained in the various constructs analyzed in Figure 3.

Figure 3

Figure 3

KYP SRA domain preferentially binds methylated DNA. (A) Mobility shift assays using increasing amounts of GST-KYP-SRA (KYP-SRA) (25, 50 and 100 nM) added to a binding reaction with either a methylated (m) or unmethylated (u) all-C double stranded oligonucleotide probe. GST-KYP-SET (KYP-SET) was added at 100 nM in the indicated lanes. Anti-GST antibodies (AB) were added to the binding reaction shown in the last lane to yield a super-shifted complex. KYP-SRA (B), KYP-SRA(E208K) (D) or KYP-SRA(S200F) (F) was bound to oligonucleotides with cytosines methylated in all sequences contexts (all-mC), or in a CG, CNG, or CNN context. In D and F the last lane is GST-KYP-SRA bound to the all-mC probe used as a positive control. (C) Competition assays were performed by addition of the indicated unlabeled DNA, added in 1000 fold excess to the all-mC probe. (E) GST-SUVH6-SRA (10 nM) was bound to the indicated probes. (G-H) GST-SUVH6-SRA (10 nM) or GST-KYP-SRA (100 nM) was bound to the methylated CNG probe with either no competitor (-), or 100x, 300x, 1000x, or 3000x unlabeled mCG or mCNG oligonucleotides. The lane indicated by fp (free probe) has no added protein.

Figure 4

Figure 4

Binding of the ORTH proteins to methylated DNA is dependent on the SRA domain. (A) Diagram of the different regions of ORTH1 or ORTH2 fused to GST and used in mobility shift assays. (B) Mobility shift assays were done with the indicated protein using either unmethylated or methylated CG probes. *The last line in each panel contains binding of GST-PNS to methylated CG super-shifted by addition of anti-GST antibody. (C) Mobility shift assays were performed with increasing amounts (35, 70, and 140 nM) of GST-PNS binding to mCG, mCNG, or mCNN. (D). Mobility shift assays with increasing amounts of GST-PNS (70, 140, and 280 nM) or GST-KYP (77, 154, and 308 nM) with methylated (Methylated) or unmethylated (U) CG. The first lane in all panels is the free probe (fp).

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