DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions.

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.

Regulatory mechanisms governing chromatin organization and function.

Nucleosomes, the basic structures used to package genetic information into chromatin, are subject to a diverse array of chemical modifications. A large number of these marks serve as interaction hubs for many nuclear proteins and provide critical structural features for protein recruitment. Dynamic deposition and removal of chromatin modifications by regulatory proteins ensure their correct deposition to the genome, which is essential for DNA replication, transcription, chromatin compaction, or DNA damage repair. The spatiotemporal regulation and maintenance of chromatin marks relies on coordinated activities of writer, eraser, and reader enzymes and often depends on complex multicomponent regulatory circuits. In recent years, the field has made enormous advances in uncovering the mechanisms that regulate chromatin modifications. Here, we discuss well-established and emerging concepts in chromatin biology ranging from cooperativity and multivalent interactions to regulatory feedback loops and increased local concentration of chromatin-modifying enzymes.

Publisher Correction: ChromID identifies the protein interactome at chromatin marks.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ChromID identifies the protein interactome at chromatin marks.

Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

Isoform-specific localization of DNMT3A regulates DNA methylation fidelity at bivalent CpG islands.

DNA methylation is a prevalent epigenetic modification involved in transcriptional regulation and essential for mammalian development. While the genome-wide distribution of this mark has been studied to great detail, the mechanisms responsible for its correct deposition, as well as the cause for its aberrant localization in cancers, have not been fully elucidated. Here, we have compared the activity of individual DNMT3A isoforms in mouse embryonic stem and neuronal progenitor cells and report that these isoforms differ in their genomic binding and DNA methylation activity at regulatory sites. We identify that the longer isoform DNMT3A1 preferentially localizes to the methylated shores of bivalent CpG island promoters in a tissue-specific manner. The isoform-specific targeting of DNMT3A1 coincides with elevated hydroxymethylcytosine (5-hmC) deposition, suggesting an involvement of this isoform in mediating turnover of DNA methylation at these sites. Through genetic deletion and rescue experiments, we demonstrate that this isoform-specific recruitment plays a role in de novo DNA methylation at CpG island shores, with potential implications on H3K27me3-mediated regulation of developmental genes.

Genome-Engineering Tools to Establish Accurate Reporter Cell Lines That Enable Identification of Therapeutic Strategies to Treat Friedreich's Ataxia.

Friedreich's ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genome-engineering tools that enable the generation of human cell lines that express the frataxin gene fused to a luciferase reporter gene from its endogenous locus. Performing a pilot high-throughput genomic screen in a newly established reporter cell line, we uncovered novel negative regulators of frataxin expression. Rational design of small-molecule inhibitors of the identified frataxin repressors and/or high-throughput screening of large siRNA or compound libraries with our system may yield treatments for Friedreich's ataxia.