The ChAHP Complex Counteracts Chromatin Looping at CTCF Sites that Emerged from SINE Expansions in Mouse.

CCCTC-binding factor (CTCF) and cohesin are key players in three-dimensional chromatin organization. The topologically associating domains (TADs) demarcated by CTCF are remarkably well conserved between species, although genome-wide CTCF binding has diverged substantially following transposon-mediated motif expansions. Therefore, the CTCF consensus motif poorly predicts TADs, and additional factors must modulate CTCF binding and subsequent TAD formation. Here, we demonstrate that the ChAHP complex (CHD4, ADNP, HP1) competes with CTCF for a common set of binding motifs. In Adnp knockout cells, novel insulated regions are formed at sites normally bound by ChAHP, whereas proximal canonical boundaries are weakened. These data reveal that CTCF-mediated loop formation is modulated by a distinct zinc-finger protein complex. Strikingly, ChAHP-bound loci are mainly situated within less diverged SINE B2 transposable elements. This implicates ChAHP in maintenance of evolutionarily conserved spatial chromatin organization by buffering novel CTCF binding sites that emerged through SINE expansions.

ChAHP2 and ChAHP control diverse retrotransposons by complementary activities.

Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1ß-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila.

Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 23-30 nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here, we show that a complex composed of Rhino, Deadlock, and Cutoff (RDC) defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC is anchored to H3K9me3-marked chromatin in part via Rhino's chromodomain. Depletion of Piwi results in loss of the RDC and small RNAs at a subset of piRNA clusters, demonstrating a feedback loop between Piwi and piRNA source loci. Intriguingly, profiles of RNA polymerase II occupancy, nascent transcription, and steady-state RNA levels reveal that the RDC licenses noncanonical transcription of dual-strand piRNA clusters. Likely, this process involves 5' end protection of nascent RNAs and suppression of transcription termination. Our data provide key insight into the regulation and evolution of piRNA clusters.

Inheritance of a Phenotypically Neutral Epimutation Evokes Gene Silencing in Later Generations.

Small RNAs trigger the formation of epialleles that are silenced across generations. Consequently, RNA-directed epimutagenesis is associated with persistent gene repression. Here, we demonstrate that small interfering RNA-induced epimutations in fission yeast are still inherited even when the silenced gene is reactivated, and descendants can reinstate the silencing phenotype that only occurred in their ancestors. This process is mediated by the deposition of a phenotypically neutral molecular mark composed of tri-methylated histone H3 lysine 9 (H3K9me3). Its stable propagation is coupled to RNAi and requires maximal binding affinity of the Clr4/Suvar39 chromodomain to H3K9me3. In wild-type cells, this mark has no visible impact on transcription but causes gene silencing if RNA polymerase-associated factor 1 complex (Paf1C) activity is impaired. In sum, our results reveal a distinct form of epigenetic memory in which cells acquire heritable, transcriptionally active epialleles that confer gene silencing upon modulation of Paf1C.

Mouse nuclear RNAi-defective 2 promotes splicing of weak 5' splice sites.

Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.

guidedNOMe-seq quantifies chromatin states at single allele resolution for hundreds of custom regions in parallel.

Since the introduction of next generation sequencing technologies, the field of epigenomics has evolved rapidly. However, most commonly used assays are enrichment-based methods and thus only semi-quantitative. Nucleosome occupancy and methylome sequencing (NOMe-seq) allows for quantitative inference of chromatin states with single locus resolution, but this requires high sequencing depth and is therefore prohibitively expensive to routinely apply to organisms with large genomes. To overcome this limitation, we introduce guidedNOMe-seq, where we combine NOMe profiling with large scale sgRNA synthesis and Cas9-mediated region-of-interest (ROI) liberation. To facilitate quantitative comparisons between multiple samples, we additionally develop an R package to standardize differential analysis of any type of NOMe-seq data. We extensively benchmark guidedNOMe-seq in a proof-of-concept study, dissecting the interplay of ChAHP and CTCF on chromatin. In summary we present a cost-effective, scalable, and customizable target enrichment extension to the existing NOMe-seq protocol allowing genome-scale quantification of nucleosome occupancy and transcription factor binding at single allele resolution.

Activity-dependent neuroprotective protein recruits HP1 and CHD4 to control lineage-specifying genes.

De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions 1 . ADNP is a putative transcription factor that is essential for embryonic development 2 . However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination 5 . Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons6,7.

The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts.

Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5' end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA-DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms.

Genetic and mechanistic diversity of piRNA 3'-end formation.

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)-the 22-30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads-are generated independently of Dicer from single-stranded precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD-a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. Formation of piRNA 3' ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3'-end formation.

Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors.

Cellular differentiation entails loss of pluripotency and gain of lineage- and cell-type-specific characteristics. Using a murine system that progresses from stem cells to lineage-committed progenitors to terminally differentiated neurons, we analyzed DNA methylation and Polycomb-mediated histone H3 methylation (H3K27me3). We show that several hundred promoters, including pluripotency and germline-specific genes, become DNA methylated in lineage-committed progenitor cells, suggesting that DNA methylation may already repress pluripotency in progenitor cells. Conversely, we detect loss and acquisition of H3K27me3 at additional targets in both progenitor and terminal states. Surprisingly, many neuron-specific genes that become activated upon terminal differentiation are Polycomb targets only in progenitor cells. Moreover, promoters marked by H3K27me3 in stem cells frequently become DNA methylated during differentiation, suggesting context-dependent crosstalk between Polycomb and DNA methylation. These data suggest a model how de novo DNA methylation and dynamic switches in Polycomb targets restrict pluripotency and define the developmental potential of progenitor cells.

Genomic prevalence of heterochromatic H3K9me2 and transcription do not discriminate pluripotent from terminally differentiated cells.

Cellular differentiation entails reprogramming of the transcriptome from a pluripotent to a unipotent fate. This process was suggested to coincide with a global increase of repressive heterochromatin, which results in a reduction of transcriptional plasticity and potential. Here we report the dynamics of the transcriptome and an abundant heterochromatic histone modification, dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal differentiation of embryonic stem cells. In contrast to the prevailing model, we find H3K9me2 to occupy over 50% of chromosomal regions already in stem cells. Marked are most genomic regions that are devoid of transcription and a subgroup of histone modifications. Importantly, no global increase occurs during differentiation, but discrete local changes of H3K9me2 particularly at genic regions can be detected. Mirroring the cell fate change, many genes show altered expression upon differentiation. Quantitative sequencing of transcripts demonstrates however that the total number of active genes is equal between stem cells and several tested differentiated cell types. Together, these findings reveal high prevalence of a heterochromatic mark in stem cells and challenge the model of low abundance of epigenetic repression and resulting global basal level transcription in stem cells. This suggests that cellular differentiation entails local rather than global changes in epigenetic repression and transcriptional activity.

Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.

Genetics and epigenetics: stability and plasticity during cellular differentiation.

Stem cells and multipotent progenitor cells face the challenge of balancing the stability and plasticity of their developmental states. Their self-renewal requires the maintenance of a defined gene-expression program, which must be stably adjusted towards a new fate upon differentiation. Recent data imply that epigenetic mechanisms can confer robustness to steady state gene expression but can also direct the terminal fate of lineage-restricted multipotent progenitor cells. Here, we review the latest models for how changes in chromatin and DNA methylation are regulated during cellular differentiation. We further propose that targets of epigenetic repression share common features in the sequences of their regulatory regions, thereby suggesting a co-evolution of epigenetic pathways and classes of cis-acting elements.

DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity.

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG-rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a(-/-) cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1alpha binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially 'rescues' the DNA methylation defect observed in G9a(-/-) cells. Taken together, these observations reveal that H3K9me3 and HP1alpha recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.

EJC-independent degradation of nonsense immunoglobulin-mu mRNA depends on 3' UTR length.

Inconsistent with prevailing models for nonsense-mediated mRNA decay (NMD) in mammals, the mRNA levels of immunoglobulin-mu (Ig-mu) genes with premature termination codons (PTCs) in the penultimate exon are still reduced by NMD when the intron furthest downstream is deleted. As in yeast, this exon junction complex-independent NMD of Ig-mu mRNAs depends on the distance between the termination codon and the poly(A) tail and suggests an evolutionarily conserved mode of PTC recognition.

Methylated DNA immunoprecipitation (MeDIP).

Methylated DNA immunoprecipitation (MeDIP) is a versatile immunocapturing approach for unbiased detection of methylated DNA. In brief, genomic DNA is randomly sheared by sonication and immunoprecipitated with a monoclonal antibody that specifically recognizes 5-methylcytidine. The resulting enrichment of methylated DNA in the immunoprecipitated fraction can be determined by PCR to assess the methylation state of individual regions. Alternatively, MeDIP can be combined with large-scale analysis using microarrays as a genome-wide experimental readout. This protocol has been applied to generate comprehensive DNA methylation profiles on a genome-wide scale in mammals and plants, and further to identify abnormally methylated genes in cancer cells.

Author Correction: H3K64 trimethylation marks heterochromatin and is dynamically remodeled during developmental reprogramming.

In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.

Distinctive features of lincRNA gene expression suggest widespread RNA-independent functions.

Eukaryotic genomes produce RNAs lacking protein-coding potential, with enigmatic roles. We integrated three approaches to study large intervening noncoding RNA (lincRNA) gene functions. First, we profiled mouse embryonic stem cells and neural precursor cells at single-cell resolution, revealing lincRNAs expressed in specific cell types, cell subpopulations, or cell cycle stages. Second, we assembled a transcriptome-wide atlas of nuclear lincRNA degradation by identifying targets of the exosome cofactor Mtr4. Third, we developed a reversible depletion system to separate the role of a lincRNA gene from that of its RNA. Our approach distinguished lincRNA loci functioning in trans from those modulating local gene expression. Some genes express stable and/or abundant lincRNAs in single cells, but many prematurely terminate transcription and produce lincRNAs rapidly degraded by the nuclear exosome. This suggests that besides RNA-dependent functions, lincRNA loci act as DNA elements or through transcription. Our integrative approach helps distinguish these mechanisms.

Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis.

In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends.

Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.

Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.