Nucleosomal asymmetry: a novel mechanism to regulate nucleosome function.

Nucleosomes constitute the fundamental building blocks of chromatin. They are comprised of DNA wrapped around a histone octamer formed of two copies each of the four core histones H2A, H2B, H3, and H4. Nucleosomal histones undergo a plethora of posttranslational modifications that regulate gene expression and other chromatin-templated processes by altering chromatin structure or by recruiting effector proteins. Given their symmetric arrangement, the sister histones within a nucleosome have commonly been considered to be equivalent and to carry the same modifications. However, it is now clear that nucleosomes can exhibit asymmetry, combining differentially modified sister histones or different variants of the same histone within a single nucleosome. Enabled by the development of novel tools that allow generating asymmetrically modified nucleosomes, recent biochemical and cell-based studies have begun to shed light on the origins and functional consequences of nucleosomal asymmetry. These studies indicate that nucleosomal asymmetry represents a novel regulatory mechanism in the establishment and functional readout of chromatin states. Asymmetry expands the combinatorial space available for setting up complex sets of histone marks at individual nucleosomes, regulating multivalent interactions with histone modifiers and readers. The resulting functional consequences of asymmetry regulate transcription, poising of developmental gene expression by bivalent chromatin, and the mechanisms by which oncohistones deregulate chromatin states in cancer. Here, we review recent progress and current challenges in uncovering the mechanisms and biological functions of nucleosomal asymmetry.

Epigenetic dynamics during capacitation of naïve human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

Chromatin remodeling of histone H3 variants underlies epigenetic inheritance of DNA methylation.

Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1 Lsh/HELLS ), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro . We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo . DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1 Dnmt1 . DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.

Detection and Quantification of Histone Methyltransferase Activity In Vitro.

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeler function.

The Isw1b chromatin-remodeling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeler. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promotes the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodeling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs.

Histone marks regulate the epithelial-to-mesenchymal transition via alternative splicing.

Histone modifications impact final splicing decisions. However, there is little evidence of the driving role of these marks in inducing cell-specific splicing changes. Using CRISPR epigenome editing tools, we show in an epithelial-to-mesenchymal cell reprogramming system (epithelial-to-mesenchymal transition [EMT]) that a single change in H3K27ac or H3K27me3 levels right at the alternatively spliced exon is necessary and sufficient to induce a splicing change capable of recapitulating important aspects of EMT, such as cell motility and invasiveness. This histone-mark-dependent splicing effect is highly dynamic and mediated by direct recruitment of the splicing regulator PTB to its RNA binding sites. These results support a role for H3K27 marks in inducing a change in the cell's phenotype via regulation of alternative splicing. We propose the dynamic nature of chromatin as a rapid and reversible mechanism to coordinate the splicing response to cell-extrinsic cues, such as induction of EMT.

H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation.

Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.

The 3' processing of antisense RNAs physically links to chromatin-based transcriptional control.

Noncoding RNA plays essential roles in transcriptional control and chromatin silencing. At Arabidopsis thaliana FLC, antisense transcription quantitatively influences transcriptional output, but the mechanism by which this occurs is still unclear. Proximal polyadenylation of the antisense transcripts by FCA, an RNA-binding protein that physically interacts with RNA 3' processing factors, reduces FLC transcription. This process genetically requires FLD, a homolog of the H3K4 demethylase LSD1. However, the mechanism linking RNA processing to FLD function had not been established. Here, we show that FLD tightly associates with LUMINIDEPENDENS (LD) and SET DOMAIN GROUP 26 (SDG26) in vivo, and, together, they prevent accumulation of monomethylated H3K4 (H3K4me1) over the FLC gene body. SDG26 interacts with the RNA 3' processing factor FY (WDR33), thus linking activities for proximal polyadenylation of the antisense transcripts to FLD/LD/SDG26-associated H3K4 demethylation. We propose this demethylation antagonizes an active transcription module, thus reducing H3K36me3 accumulation and increasing H3K27me3. Consistent with this view, we show that Polycomb Repressive Complex 2 (PRC2) silencing is genetically required by FCA to repress FLC Overall, our work provides insights into RNA-mediated chromatin silencing.

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.

Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin.

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines1,2. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm3,4. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. 5), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

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.

Borealin-nucleosome interaction secures chromosome association of the chromosomal passenger complex.

Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.

R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes.

R-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.

Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma.

A methionine substitution at lysine-27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits polycomb repressive complex 2 (PRC2) in a dominant-negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found that this interaction on chromatin is transient, with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-containing chromatin, suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to H3K27M, leading to the failure to spread H3K27me from PRC2 recruitment sites and consequently abrogating PRC2's ability to establish H3K27me2-3 repressive chromatin domains. In turn, levels of polycomb antagonists such as H3K36me2 are elevated, suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.

In Vitro Assays to Measure Histone Methyltransferase Activity Using Different Chromatin Substrates.

In vitro histone modification (HM) assays are used to characterize the activity of chromatin-modifying enzymes. These assays provide information regarding the modification sites on histones, the product specificity, and the impact of other histone or nucleotide modifications on enzyme activity. In particular, histone methyltransferase (HMT) assays have been instrumental in elucidating the activity and site specificity of many plant HMT enzymes. In this chapter, we describe a general protocol that can be used to perform HMT assays using different chromatin substrates, detection methods, and enzymes directly purified from plant material or heterologous sources.

Chloromethyl-triazole: a new motif for site-selective pseudo-acylation of proteins.

Rapid, site-selective modification of cysteine residues with chloromethyl-triazole derivatives generates pseudo-acyl sLys motifs, mimicking important post-translational modifications. Near-native biotinylation of peptide and protein substrates is shown to be site-selective and modified histone H4 retains functional activity.

Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.