Light-Activation of DNA-Methyltransferases

Abstract

5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.

Keywords: DNA methyltransferases; epigenetics; genetic code expansion; optochemical biology.

Figure 1

Light‐control of DNA methyltransferase catalysis. a) Mechanism of DNMT‐catalyzed cytosine methylation at C5. SAM: S‐adenosylmethionine; SAH: S‐adenosylhomocysteine. b) Crystal structure of human DNMT3a active site with catalytic C710 trapped with the cytosine analog zebularine (Z, pdb 6F57 ^[11] ). c) Structure and light‐deprotection of photocaged cysteine 1. d) Domain and crystal structure (pdb 6F57 ^[11] ) overviews of pcDNMT3a3L with position of catalytic C710→1 mutation for light‐activation. NLS: nuclear localization sequence. HA: hemagglutinin tag. e) Incorporation of 1 at pcDNMT3a3L amber codon (C710TAG) in HCT116 DKO cells analyzed by anti‐HA immunostaining and flow cytometry (FCM). Error bars show standard deviations from three independent biological replicates. f) Anti‐5mC immunostainings and FCM analysis of HCT116 DKO cells expressing pcDNMT3a3L 24 h after light or no light irradiation. Error bars show standard deviations from three independent biological replicates.

Figure 2

In vivo measurements of DNA methylation catalysis by pcDNMT3a3L with frequent cancer‐associated DNMT3a mutations using light‐control. a) Domain structure of pcDNMT3a3L with conserved DNMT motifs I–X and mutation positions analyzed in this study. Catalytic loop in green, TRD loop in yellow, part of homodimer interface in orange. b) Crystal structure of human DNMT3a active site bound to DNA (pdb 6F57 ^[11] ) with mutation positions analyzed by light activation shown as blue sticks. DNMT3L in pink, other color codes as in (a). c) Activity studies of pcDNMT3a3L cancer mutants conducted by anti‐5mC immunostainings and FCM. Measurements were conducted 24 h after light or no light irradiation. At this point, 5mC formation has not reached saturation for the wt (e.g., compare to R771G reaching even higher 5mC level). Error bars show standard deviations from three independent biological replicates.

Figure 3

Light‐controlled, targeted methylation of SATIII loci. a) Domain structure of SATIII‐pcDNMT construct. b) Images of heat‐stressed HEK293T cells expressing SATIII‐pcDNMT immunostained with anti‐HA and anti‐HSF1 antibodies. Scale bar is 5 μm. c) Kinetics of SATIII methylation with SATIII‐pcDNMT after light irradiation and no light controls. 5mC was quantified by bisulfite PCR and Illumina amplicon deep sequencing. Error bars show standard deviations from three independent biological replicates. UT=untransfected + light. d) Methylation of SATIII and two off‐target loci by SATIII‐pcDNMT 24 h after irradiation analyzed by Sanger sequencing of bisulfite PCR products. gDNA sequence is shown below with Cs bold and CpG underlined. e) Methylation of the BRCA1 and p16 loci of (d) with TALE‐DNMT constructs designed to specifically target the BRCA1 and p16 loci, respectively. f) Light dosage dependence of SATIII methylation in experiments conducted as in (c). 5mC was quantified by bisulfite PCR and pyrosequencing. Error bars show standard deviations from three independent biological replicates. UT=untransfected.

Figure 4

Light‐activated transcriptome regulation by pcDNMT3a in HEK293T cells. Gene expression patterns from mRNA‐Seq 0 h, 4 h and 8 h after light‐activation of pcDNMT3a. Two exemplary groups of differentially expressed genes with distinct expression patterns are shown (independent biological duplicates, full image in Figure S18) as log2‐fold change against average expression over all conditions; differential light‐dependent expression at a false discovery rate of <0.05.