Pericentromeric Satellite III transcripts induce etoposide resistance.

Non-coding RNA from pericentromeric satellite repeats are involved in stress-dependent splicing processes, maintenance of heterochromatin, and are required to protect genome stability. Here we show that the long non-coding satellite III RNA (SatIII) generates resistance against the topoisomerase IIa (TOP2A) inhibitor etoposide in lung cancer. Because heat shock conditions (HS) protect cells against the toxicity of etoposide, and SatIII is significantly induced under HS, we hypothesized that the protective effect could be traced back to SatIII. Using genome methylation profiles of patient-derived xenograft mouse models we show that the epigenetic modification of the SatIII DNA locus and the resulting SatIII expression predict chemotherapy resistance. In response to stress, SatIII recruits TOP2A to nuclear stress bodies, which protects TOP2A from a complex formation with etoposide and results in decreased DNA damage after treatment. We show that BRD4 inhibitors reduce the expression of SatIII, restoring etoposide sensitivity.

Light-Activation of DNA-Methyltransferases.

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.

Designer Receptors for Nucleotide-Resolution Analysis of Genomic 5-Methylcytosine by Cellular Imaging.

We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.