SummaryChromatin organization is disrupted genome-wide during DNA replication. On newly synthesized DNA, nucleosomes are assembled from new naive histones and old modified histones. It remains unknown whether the landscape of histone post-translational modifications (PTMs) is faithfully copied during DNA replication or the epigenome is perturbed. Here we develop chromatin occupancy after replication (ChOR-seq) to determine histone PTM occupancy immediately after DNA replication and across the cell cycle. We show that H3K4me3, H3K36me3, H3K79me3, and H3K27me3 positional information is reproduced with high accuracy on newly synthesized DNA through histone recycling. Quantitative ChOR-seq reveals that de novo methylation to restore H3K4me3 and H3K27me3 levels occurs across the cell cycle with mark- and locus-specific kinetics. Collectively, this demonstrates that accurate parental histone recycling preserves positional information and allows PTM transmission to daughter cells while modification of new histones gives rise to complex epigenome fluctuations across the cell cycle that could underlie cell-to-cell heterogeneity.

RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA (dsRNA) to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the dsRNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically posttranscriptional gene silencing (PTGS) and chromatin-dependent gene silencing 1 . Although endogenous small RNAs play critical roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymeraseassociated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model where Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing (RITS) complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of Reprints and permissions information is available at www.nature.com/reprints. A patent application has been filed.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsCorrespondence and requests for materials should be addressed to M.B. (marc.buehler@fmi.ch). * Correspondence to: marc.buehler@fmi.ch. Author Contributions Y.S., K.M.K., V.F., and J.B. generated strains and performed experiments; Y.S. performed the sms screen; genome-wide small RNA and gene expression data was analyzed by K.M.K.; M.B.S. designed and performed the computational analysis of the mutant genome resequencing data; M.B. designed experiments and prepared the manuscript. All authors discussed the results and commented on the manuscript.

SummaryFaithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1's phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3–H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3–H4 supply. We determine the structures of a sNASP dimer, a complex of a sNASP dimer with two H3 α3 peptides, and the sNASP–H3–H4–ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 α3 helix and the H3 αN region, respectively. Functional studies demonstrate the H3 αN-interaction represents the major binding mode of NASP in cells and shielding of the H3 αN region by NASP is essential in maintaining the H3–H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3–H4 chaperone in guarding histone homeostasis.

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3–H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3–H4 supply. We determine the structures of a sNASP dimer, a complex of sNASP with an H3 α3 peptide, and the sNASP–H3–H4–ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 α3 helix and the H3 αN region, respectively. Functional studies demonstrate the H3 αN-interaction represents the major binding mode of NASP in cells and shielding of the H3 αN region by NASP is essential in maintaining the H3–H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3–H4 chaperone in guarding histone homeostasis.

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Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.