ABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , DNA Methylation , Histones , Nucleosomes , Chromatin Assembly and Disassembly , DNA , DNA Modification Methylases , Epigenesis, Genetic , Histones/genetics , Nucleosomes/genetics , Semen , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolismABSTRACT
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.
ABSTRACT
Recent studies have indicated that nucleosome turnover is rapid, occurring several times per cell cycle. To access the effect of nucleosome turnover on the epigenetic landscape, we investigated H3K79 methylation, which is produced by a single methyltransferase (Dot1l) with no known demethylase. Using chemical-induced proximity (CIP), we find that the valency of H3K79 methylation (mono-, di-, and tri-) is determined by nucleosome turnover rates. Furthermore, propagation of this mark is predicted by nucleosome turnover simulations over the genome and accounts for the asymmetric distribution of H3K79me toward the transcriptional unit. More broadly, a meta-analysis of other conserved histone modifications demonstrates that nucleosome turnover models predict both valency and chromosomal propagation of methylation marks. Based on data from worms, flies, and mice, we propose that the turnover of modified nucleosomes is a general means of propagation of epigenetic marks and a determinant of methylation valence.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Genome , Histones/metabolism , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Chromatin Assembly and Disassembly , Computer Simulation , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , HEK293 Cells , Histone-Lysine N-Methyltransferase , Histones/genetics , Humans , Jurkat Cells , Kinetics , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Models, Genetic , Monte Carlo Method , Nucleosomes/geneticsABSTRACT
Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , Epigenesis, Genetic , Gene Editing , CRISPR-Cas Systems , Gene Expression Regulation , Gene Silencing , HEK293 Cells , Heterochromatin/metabolism , Humans , Polycomb-Group Proteins/metabolism , Promoter Regions, GeneticABSTRACT
The resolution and formation of facultative heterochromatin are essential for development, reprogramming, and oncogenesis. The mechanisms underlying these changes are poorly understood owing to the difficulty of studying heterochromatin dynamics and structure in vivo. We devised an in vivo approach to investigate these mechanisms and found that topoisomerase II (TOP2), but not TOP1, synergizes with BAF (mSWI/SNF) ATP-dependent chromatin remodeling complexes genome-wide to resolve facultative heterochromatin to accessible chromatin independent of transcription. This indicates that changes in DNA topology that take place through (de-)catenation rather than the release of torsional stress through swiveling are necessary for heterochromatin resolution. TOP2 and BAF cooperate to recruit pluripotency factors, which explains some of the instructive roles of BAF complexes. Unexpectedly, we found that TOP2 also plays a role in the re-formation of facultative heterochromatin; this finding suggests that facultative heterochromatin and accessible chromatin exist at different states of catenation or other topologies, which might be critical to their structures.
Subject(s)
Chromatin Assembly and Disassembly , DNA Topoisomerases, Type II/metabolism , Heterochromatin/metabolism , Animals , Chromatin Immunoprecipitation , DNA/metabolism , Electrophoresis, Agar Gel , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Genetic Loci , Mice , Protein Binding/drug effects , Protein Binding/genetics , Sirolimus/pharmacology , Time Factors , Transcription Initiation Site , Transcription, Genetic/drug effectsABSTRACT
Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.
Subject(s)
Adenosine Triphosphatases/genetics , Cell Cycle Proteins/genetics , Chromatin/genetics , DNA Helicases/genetics , Nuclear Proteins/genetics , Point Mutation/genetics , Polycomb-Group Proteins/genetics , Transcription Factors/genetics , Adenosine Triphosphate/genetics , Animals , Chromatin Assembly and Disassembly/genetics , DNA-Binding Proteins/genetics , Mice , Neurodevelopmental Disorders/geneticsABSTRACT
The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.
Subject(s)
Carcinogenesis/genetics , DNA-Binding Proteins/genetics , Heterochromatin/genetics , Nuclear Proteins/genetics , Polycomb-Group Proteins/genetics , Chromatin/genetics , DNA Replication/genetics , Epigenesis, Genetic/genetics , Humans , Mutation/genetics , RNA Polymerase II/genetics , Transcription, Genetic/geneticsABSTRACT
In mammals and plants, parental genomic imprinting restricts the expression of specific loci to one parental allele. Imprinting in mammals relies on sex-dependent de novo deposition of DNA methylation during gametogenesis but a comparable mechanism was not shown in plants. Rather, paternal silencing by the maintenance DNA methyltransferase 1 (MET1) and maternal activation by the DNA demethylase DEMETER (DME) cause maternal expression. However, genome-wide studies suggested other DNA methylation-dependent imprinting mechanisms. Here, we show that de novo RNA-directed DNA methylation (RdDM) regulates imprinting at specific loci expressed in endosperm. RdDM in somatic tissues is required to silence expression of the paternal allele. By contrast, the repression of RdDM in female gametes participates with or without DME requirement in the activation of the maternal allele. The contrasted activity of DNA methylation between male and female gametes appears sufficient to prime imprinted maternal expression. After fertilization, MET1 maintains differential expression between the parental alleles. RdDM depends on small interfering RNAs (siRNAs). The involvement of RdDM in imprinting supports the idea that sources of siRNAs such as transposons and de novo DNA methylation were recruited in a convergent manner in plants and mammals in the evolutionary process leading to selection of imprinted loci.
Subject(s)
Arabidopsis/genetics , DNA Methylation , DNA, Plant , Gene Expression Regulation, Plant , Genomic Imprinting , Arabidopsis/metabolism , Endosperm , RNA, Plant , RNA, Small Interfering/metabolismABSTRACT
Progress in studying epigenetic reprogramming in plants has been impeded by the difficulty in obtaining tissue for analysis. Now, using a combination of fluorescent reporters and translational fusions, a new study sheds some light on this process.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , DNA Methylation , Methyltransferases/metabolismABSTRACT
BACKGROUND: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. RESULTS: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. CONCLUSIONS: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.
ABSTRACT
Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.
Subject(s)
Arabidopsis/genetics , DNA Methylation , Epigenesis, Genetic , Pollen/genetics , RNA, Plant/genetics , RNA, Small Interfering/genetics , Animals , Arabidopsis/growth & development , DNA Transposable Elements , Mammals/genetics , RNA, Plant/metabolism , RNA, Small Interfering/metabolism , Seeds/genetics , Seeds/metabolismABSTRACT
The movement of mobile small RNA signals between cells has garnered much interest over the last few years, and has recently been extended to germ cells during gamete development. Focusing on plants, we review mobile RNA signals that arise following reprogramming in the germline, and their effect on transposable element silencing on the one hand and on meiotic and apomictic germ cell fate on the other. A potential role for reprogramming and small RNA in hybrid formation and speciation is proposed.
Subject(s)
Arabidopsis/genetics , Genome, Plant , Germ Cells, Plant/metabolism , RNA, Plant/genetics , RNA, Small Interfering/genetics , Animals , Humans , RNA, Small Interfering/biosynthesisABSTRACT
Alternative splicing is a powerful mechanism affording extensive proteomic and regulatory diversity from a limited repertoire of genes. However, the extent to which alternative splicing has contributed to the evolution of primate species-specific characteristics has not been assessed previously. Using comparative genomics and quantitative microarray profiling, we performed the first global analysis of alternative splicing differences between humans and chimpanzees. Surprisingly, 6%-8% of profiled orthologous exons display pronounced splicing level differences in the corresponding tissues from the two species. Little overlap is observed between the genes associated with alternative splicing differences and the genes that display steady-state transcript level differences, indicating that these layers of regulation have evolved rapidly to affect distinct subsets of genes in humans and chimpanzees. The alternative splicing differences we detected are predicted to affect diverse functions including gene expression, signal transduction, cell death, immune defense, and susceptibility to diseases. Differences in expression at the protein level of the major splice variant of Glutathione S-transferase omega-2 (GSTO2), which functions in the protection against oxidative stress and is associated with human aging-related diseases, suggests that this enzyme is less active in human cells compared with chimpanzee cells. The results of this study thus support an important role for alternative splicing in establishing differences between humans and chimpanzees.
Subject(s)
Alternative Splicing/genetics , Genome, Human , Pan troglodytes/genetics , Animals , Evolution, Molecular , Exons , Expressed Sequence Tags/chemistry , Gene Expression Profiling , Gene Expression Regulation, Enzymologic/genetics , Genetic Variation , Genomics , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , HeLa Cells , Humans , Introns , Isoenzymes/genetics , Oligonucleotide Array Sequence Analysis , Plasmids , RNA, Messenger/genetics , Species Specificity , TransfectionABSTRACT
The polarized glandular organization of epithelial cells is frequently lost during development of carcinoma. However, the specific oncogene targets responsible for polarity disruption have not been identified. Here, we demonstrate that activation of ErbB2 disrupts apical-basal polarity by associating with Par6-aPKC, components of the Par polarity complex. Inhibition of interaction between Par6 and aPKC blocked the ability of ErbB2 to disrupt the acinar organization of breast epithelia and to protect cells from apoptosis but was not required for cell proliferation. Therefore, oncogenes target polarity proteins to disrupt glandular organization and protect cells from apoptotic death during development of carcinoma.
Subject(s)
Carrier Proteins/metabolism , Cell Proliferation , Epithelial Cells/metabolism , Protein Kinase C/metabolism , Receptor, ErbB-2/metabolism , Animals , Apoptosis/physiology , Cell Cycle/physiology , Cell Line , Cell Polarity/physiology , Epithelial Cells/cytology , Gene Expression , Immunoblotting , Immunoprecipitation , Microscopy, Fluorescence , Protein Binding , Receptor, ErbB-2/genetics , Signal Transduction/physiologyABSTRACT
Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain. As predicted, the trans form of the chromophore destabilizes the helical structure of the coiled-coil region of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis photoisomerization of the chromophore increases helical content and substantially enhances DNA binding. The system is observed to be readily reversible; thermal relaxation of the chromophore to the trans state and concomitant dissociation of the protein-DNA complex occurs with tau(1/2) approximately 10 min at 37 degrees C. It appears that conformational dynamics in the zipper domain make the transition state for isomerization readily available so that retention of reversible switching is observed.