The N-terminus of histone H3 is required for de novo DNA methylation in chromatin

Abstract

DNA methylation and histone modification are two major epigenetic pathways that interplay to regulate transcriptional activity and other genome functions. Dnmt3L is a regulatory factor for the de novo DNA methyltransferases Dnmt3a and Dnmt3b. Although recent biochemical studies have revealed that Dnmt3L binds to the tail of histone H3 with unmethylated lysine 4 in vitro, the requirement of chromatin components for DNA methylation has not been examined, and functional evidence for the connection of histone tails to DNA methylation is still lacking. Here, we used the budding yeast Saccharomyces cerevisiae as a model system to investigate the chromatin determinants of DNA methylation through ectopic expression of murine Dnmt3a and Dnmt3L. We found that the N terminus of histone H3 tail is required for de novo methylation, while the central part encompassing lysines 9 and 27, as well as the H4 tail are dispensable. DNA methylation occurs predominantly in heterochromatin regions lacking H3K4 methylation. In mutant strains depleted of H3K4 methylation, the DNA methylation level increased 5-fold. The methylation activity of Dnmt3a largely depends on the Dnmt3L's PHD domain recognizing the histone H3 tail with unmethylated lysine 4. Functional analysis of Dnmt3L in mouse ES cells confirmed that the chromatin-recognition ability of Dnmt3L's PHD domain is indeed required for efficient methylation at the promoter of the endogenous Dnmt3L gene. These findings establish the N terminus of histone H3 tail with an unmethylated lysine 4 as a chromatin determinant for DNA methylation.

Fig. 1.

Yeast genomic DNA methylation by coexpressed Dnmt3a-Dnmt3L. (A) Western detection of HA-tagged Dnmt3a and Flag-tagged Dnmt3L expressed in different yeast strains upon galactose induction. Each strain was transformed with one or both of the expression constructs (+) or empty vectors (−) as indicated on the Top. (B) HPLC measurement (see Materials and Methods) of the methylcytosine content in genomic DNA isolated from the various strains. The bar graph shows the average percentages of methylcytosines in total cytosines and standard deviation among three clones for each strain.

Fig. 2.

Requirement of the N-terminal of histone H3 tail for DNA methylation. (A) Western detection of Flag-tagged Dnmt3a and Dnmt3L proteins in yeast strains carrying mutant histone H3 or H4. (B) Methylcytosine contents in histone mutant strains coexpressing Dnmt3a and Dnmt3L. For each histone mutant, three individual clones were analyzed. (C) MNase digestion assay of chromatin structure in strains with wild-type and mutant histones. Permeabilized nuclei were isolated from cells grown at logarithmic phase, and treated with MNase at increasing concentrations (0, 50, 100, 200, and 400 U/mL).

Fig. 3.

Increased DNA methylation in yeast depleted of histone H3 lysine K4 methylation. (A) Western analysis for H3K4 mono-, di-, and trimethylation in strains of set1Δ, swd1Δ, swd3Δ, and spp1Δ. Blotting with antibodies against unmodified H3 and actin served as loading controls. (B) Methylcytosine contents in various strains expressing Dnmt3a alone or together with Dnmt3L. (C) Western analysis for H3K4 mono-, di-, and trimethylation in set1Δ strains expressing reintroduced WT and mutant Set1 protein. (D) Methylcytosine contents in set1Δ strains expressing reintroduced wild-type and mutant Set1 protein.

Fig. 4.

Distribution of DNA methylation at genomic sequences associated with unmethylated H3K4. (A) Schematic diagrams of six yeast genomic loci chosen for DNA methylation analysis. Regions for chromatin-immunoprecipitation (ChIP) assay and bisulfite-based DNA methylation analysis are indicated. (B) ChIP with anti-H3K4 trimethylation antibody to examine the histone methylation state in the regions chosen for analysis. (C) COBRA DNA methylation analysis of six representative loci in wild-type and set1Δ strains. TaqI digestion of a PCR fragment into smaller fragments reflects full or partial methylation (me) in the region analyzed. Resistance to the digestion represents an unmethylated (un) state. (D) Bisulfite sequencing profiles of DNA methylation of four representative loci. CpG sequences are shown with filled (methylated) and open (unmethylated) circles.

Fig. 5.

Functional dependence of Dnmt3L on binding to unmethylated histone tails. (A) Western detection of Flag-tagged Dnmt3L PHD-mutants coexpressed with HA-Dnmt3a in yeast. (B) Peptide binding assay for the Dnmt3L PHD mutant proteins purified from yeast. Note that only the wild-type Dnmt3L control could bind to the H3 peptide that lacked modification at lysine 4. WDR5 (WD repeat domain 5) known to bind H3 terminus with unmethylated arginine 2 (40) was used as a control. (C) Requirement of Dnmt3L's PHD function to promote the Dnmt3a methylation activity.

Fig. 6.

Function of Dnmt3L's PHD in de novo methylation at the Dnmt3L promoter in differentiating mouse embryonic stem cells. (A) Western detection of the Dnmt3L PHD mutant proteins expressed in Dnmt3L-knockout ES cells. Flag-tagged Dnmt3L wild-type and mutants with substitutions D124A, I141W and PHD deletion were introduced into Dnmt3L-knockout ES cells to generate stable cell lines. Wild-type (WT) and Dnmt3L knockout (KO) ES cell lines were used as controls. GAPDH was used for loading normalization. (B) COBRA analysis of the methylation level at the Dnmt3L promoter upon in vitro differentiation of ES cells. PCR fragments amplified from bisulfite-treated genomic DNA were digested with TaqI. The relative amount of DNA sensitive and resistant to the digestion reflects methylated (me) and unmethylated (un) states at the TaqI sites examined. (C) Bisulfite sequencing profiles of DNA methylation of the Dnmt3L promoter in differentiated ES cells. CpG sequences are shown with filled (methylated) and open (unmethylated) circles. The numbers above the profiles show the percentages of methylated CpGs. Arrows below indicate the CpG dinucleotides within the TaqI recognition sequence.