RESUMEN
Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.
Asunto(s)
Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona , Metilación de ADN , Proteínas Fúngicas , Heterocromatina , Histonas , Heterocromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Histonas/metabolismo , Histonas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Unión Proteica , Neurospora crassa/genética , Neurospora crassa/metabolismoRESUMEN
In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.
Asunto(s)
Proteínas Fúngicas , Silenciador del Gen , Heterocromatina , Histonas , Histonas/metabolismo , Histonas/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Metilación , Regulación Fúngica de la Expresión Génica , Complejo Represivo Polycomb 2/metabolismo , Complejo Represivo Polycomb 2/genética , Nucleosomas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas del Grupo Polycomb/genética , Lisina/metabolismoRESUMEN
The highly and slightly condensed forms of chromatin, heterochromatin and euchromatin, respectively, segregate in the cell nucleus. Heterochromatin is more abundant in the nucleus periphery. Here we study the mechanism of heterochromatin segregation by modeling interphase chromosomes as diblock ring copolymers confined in a rigid spherical shell using molecular dynamics simulations. In our model, heterochromatin and euchromatin are distinguished by their bending stiffnesses only, while an interaction potential between the spherical shell and chromatin is used to model lamin-associated proteins. Our simulations indicate that in the absence of attractive interactions between the nuclear shell and the chromatin, most heterochromatin segregates towards the nuclear interior due to the depletion of less flexible heterochromatin segments from the nuclear periphery. This inverted chromatin distribution,which is opposite to the conventional case with heterochromatin dominating at the periphery, is in accord with experimental observations in rod cells. This "inversion" is also found to be independent of the heterochromatin concentration and chromosome number. The chromatin distribution at the periphery found in vivo can be recovered by further increasing the bending stiffness of heterochromatin segments or by turning on attractive interactions between the nuclear shell and heterochromatin. Our results indicate that the bending stiffness of chromatin could be a contributor to chromosome organization along with differential effects of HP1α-driven phase segregation and of loop extruders and interactions with the nuclear envelope and topological constraints.
Asunto(s)
Núcleo Celular , Cromatina , Heterocromatina , Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Cromatina/metabolismo , Simulación de Dinámica Molecular , Eucromatina/metabolismoRESUMEN
Heterochromatin de-condensation in companion gametic cells is conserved in both plants and animals. In plants, microspore undergoes asymmetric pollen mitosis (PMI) to produce a vegetative cell (VC) and a generative cell (GC). Subsequently, the GC undergoes pollen mitosis (PMII) to produce two sperm cells (SC). Consistent with heterochromatin de-condensation in the VC, H3K9me2, a heterochromatin mark, is barely detected in VC. However, how H3K9me2 is differentially regulated during pollen mitosis remains unclear. Here, we show that H3K9me2 is gradually evicted from the VC since PMI but remain unchanged in the GC and SC. ARID1, a pollen-specific transcription factor that facilitates PMII, promotes H3K9me2 maintenance in the GC/SC but slows down its eviction in the VC. The genomic targets of ARID1 mostly overlaps with H3K9me2 loci, and ARID1 recruits H3K9 methyltransferase SUVH6. Our results uncover that differential pattern of H3K9me2 between two cell types is regulated by ARID1 during pollen mitosis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Histonas , Mitosis , Polen , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Heterocromatina/metabolismo , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Histonas/genética , Metilación , Polen/metabolismo , Polen/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
CDCA7, encoding a protein with a carboxyl-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts a unique zinc-binding structure that recognizes a CpG dyad in a non-B DNA formed by two sequence motifs. CDCA7, but not ICF mutants, preferentially binds the non-B DNA with strand-specific CpG hemi-methylation. The unmethylated sequence motif is highly enriched at centromeres of human chromosomes, whereas the methylated motif is distributed throughout the genome. At S phase, CDCA7, but not ICF mutants, is concentrated in constitutive heterochromatin foci, and the formation of such foci can be inhibited by exogenous hemi-methylated non-B DNA bound by the CRD. Binding of the non-B DNA formed in juxtacentromeric regions during DNA replication provides a mechanism by which CDCA7 controls the specificity of DNA methylation.
Asunto(s)
Centrómero , Islas de CpG , Metilación de ADN , Síndromes de Inmunodeficiencia , Enfermedades de Inmunodeficiencia Primaria , Unión Proteica , Humanos , Enfermedades de Inmunodeficiencia Primaria/metabolismo , Enfermedades de Inmunodeficiencia Primaria/genética , Síndromes de Inmunodeficiencia/metabolismo , Síndromes de Inmunodeficiencia/genética , Centrómero/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Dominios Proteicos , ADN/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Mutación , Heterocromatina/metabolismo , Heterocromatina/genética , Cara/anomalías , Proteínas NuclearesRESUMEN
Epigenetic mechanisms enable cells to develop novel adaptive phenotypes without altering their genetic blueprint. Recent studies show histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), can be redistributed to establish adaptive phenotypes. We developed a precision-engineered genetic approach to trigger heterochromatin misregulation on-demand in fission yeast. This enabled us to trace genome-scale RNA and H3K9me changes over time in long-term, continuous cultures. Adaptive H3K9me establishes over remarkably slow timescales relative to the initiating stress. We captured dynamic H3K9me redistribution events which depend on an RNA binding complex MTREC, ultimately leading to cells converging on an optimal adaptive solution. Upon stress removal, cells relax to new transcriptional and chromatin states, establishing memory that is tunable and primed for future adaptive epigenetic responses. Collectively, we identify the slow kinetics of epigenetic adaptation that allow cells to discover and heritably encode novel adaptive solutions, with implications for drug resistance and response to infection.
Asunto(s)
Epigénesis Genética , Heterocromatina , Histonas , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Heterocromatina/metabolismo , Heterocromatina/genética , Histonas/metabolismo , Histonas/genética , Adaptación Fisiológica/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Regulación Fúngica de la Expresión Génica , MetilaciónRESUMEN
In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.
Asunto(s)
Ensamble y Desensamble de Cromatina , Silenciador del Gen , Neurospora crassa , Transcripción Genética , Neurospora crassa/genética , Neurospora crassa/metabolismo , Cromatina/metabolismo , Cromatina/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Nucleosomas , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Animales , Caenorhabditis elegans/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Cromatina/metabolismo , Transcripción Genética , Regulación de la Expresión Génica/genética , Heterocromatina/genética , Heterocromatina/metabolismo , ARN de Interacción con PiwiRESUMEN
Aging involves the deterioration of organismal function, leading to the emergence of multiple pathologies. Environmental stimuli, including lifestyle, can influence the trajectory of this process and may be used as tools in the pursuit of healthy aging. To evaluate the role of epigenetic mechanisms in this context, we have generated bulk tissue and single cell multi-omic maps of the male mouse dorsal hippocampus in young and old animals exposed to environmental stimulation in the form of enriched environments. We present a molecular atlas of the aging process, highlighting two distinct axes, related to inflammation and to the dysregulation of mRNA metabolism, at the functional RNA and protein level. Additionally, we report the alteration of heterochromatin domains, including the loss of bivalent chromatin and the uncovering of a heterochromatin-switch phenomenon whereby constitutive heterochromatin loss is partially mitigated through gains in facultative heterochromatin. Notably, we observed the multi-omic reversal of a great number of aging-associated alterations in the context of environmental enrichment, which was particularly linked to glial and oligodendrocyte pathways. In conclusion, our work describes the epigenomic landscape of environmental stimulation in the context of aging and reveals how lifestyle intervention can lead to the multi-layered reversal of aging-associated decline.
Asunto(s)
Envejecimiento , Epigénesis Genética , Heterocromatina , Hipocampo , Animales , Hipocampo/metabolismo , Envejecimiento/genética , Masculino , Ratones , Heterocromatina/metabolismo , Heterocromatina/genética , Ratones Endogámicos C57BL , Ambiente , ARN Mensajero/metabolismo , ARN Mensajero/genética , Análisis de la Célula IndividualRESUMEN
In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.
Asunto(s)
Epigenoma , Histonas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Histonas/metabolismo , Histonas/genética , Epigénesis Genética , Genoma de Planta , Cromatina/metabolismo , Cromatina/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Heterocromatina/metabolismo , Heterocromatina/genética , Código de Histonas/genéticaRESUMEN
Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation1-3. However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear4, as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences2,5. Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates.
Asunto(s)
Centrómero , Evolución Molecular , Heterocromatina , N-Metiltransferasa de Histona-Lisina , Histonas , Motivos de Nucleótidos , Animales , Humanos , Ratones , Centrómero/genética , Centrómero/metabolismo , Pollos , Homólogo de la Proteína Chromobox 5 , Silenciador del Gen , Heterocromatina/metabolismo , Heterocromatina/química , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/química , Histonas/metabolismo , Histonas/química , Anfioxos , Metilación , Petromyzon , Proteínas Represoras/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Serpientes , Xenopus laevis , Pez Cebra , Dedos de ZincRESUMEN
DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.
Asunto(s)
ADN-Topoisomerasas de Tipo II , Heterocromatina , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Heterocromatina/metabolismo , Animales , Inhibidores de Topoisomerasa II/farmacología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Replicación del ADN , ADN Superhelicoidal/metabolismo , ADN Superhelicoidal/química , Humanos , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN/metabolismo , ADN/química , InterfaseRESUMEN
Pericentromeric heterochromatin is mainly composed of satellite DNA sequences. Although being historically associated with transcriptional repression, some pericentromeric satellite DNA sequences are transcribed. The transcription events of pericentromeric satellite sequences occur in highly flexible biological contexts. Hence, the apparent randomness of pericentromeric satellite transcription incites the discussion about the attribution of biological functions. However, pericentromeric satellite RNAs have clear roles in the organization of nuclear structure. Silencing pericentromeric heterochromatin depends on pericentromeric satellite RNAs, that, in a feedback mechanism, contribute to the repression of pericentromeric heterochromatin. Moreover, pericentromeric satellite RNAs can also act as scaffolding molecules in condensate subnuclear structures (e.g., nuclear stress bodies). Since the formation/dissociation of nuclear condensates provides cell adaptability, pericentromeric satellite RNAs can be an epigenetic platform for regulating (sub)nuclear structure. We review current knowledge about pericentromeric satellite RNAs that, irrespective of the meaning of biological function, should be functionally addressed in regular and disease settings. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA in Disease and Development > RNA in Disease.
Asunto(s)
Heterocromatina , Satélite de ARN , Satélite de ARN/metabolismo , Satélite de ARN/genética , Humanos , Heterocromatina/metabolismo , Heterocromatina/genética , Animales , Núcleo Celular/metabolismo , Núcleo Celular/genética , Centrómero/metabolismo , Centrómero/genética , ADN Satélite/metabolismo , ADN Satélite/genéticaRESUMEN
Heterochromatic condensates (chromocenters) are critical for maintaining the silencing of heterochromatin. It is therefore puzzling that the presence of chromocenters is variable across plant species. Here we reveal that variations in the plant heterochromatin protein ADCP1 confer a diversity in chromocenter formation via phase separation. ADCP1 physically interacts with the high mobility group protein HMGA to form a complex and mediates heterochromatin condensation by multivalent interactions. The loss of intrinsically disordered regions (IDRs) in ADCP1 homologues during evolution has led to the absence of prominent chromocenter formation in various plant species, and introduction of IDR-containing ADCP1 with HMGA promotes heterochromatin condensation and retrotransposon silencing. Moreover, plants in the Cucurbitaceae group have evolved an IDR-containing chimaera of ADCP1 and HMGA, which remarkably enables formation of chromocenters. Together, our work uncovers a coevolved mechanism of phase separation in packing heterochromatin and silencing retrotransposons.
Asunto(s)
Silenciador del Gen , Heterocromatina , Retroelementos , Heterocromatina/metabolismo , Heterocromatina/genética , Retroelementos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Evolución MolecularRESUMEN
Viroids are pathogenic noncoding RNAs that completely rely on their host molecular machinery to accomplish their life cycle. Several interactions between viroids and their host molecular machinery have been identified, including interference with epigenetic mechanisms such as DNA methylation. Despite this, whether viroids influence changes in other epigenetic marks such as histone modifications remained unknown. Epigenetic regulation is particularly important during pathogenesis processes because it might be a key regulator of the dynamism of the defense response. Here we have analyzed the changes taking place in Cucumis sativus (cucumber) facultative and constitutive heterochromatin during hop stunt viroid (HSVd) infection using chromatin immunoprecipitation (ChIP) of the two main heterochromatic marks: H3K9me2 and H3K27me3. We find that HSVd infection is associated with changes in both H3K27me3 and H3K9me2, with a tendency to decrease the levels of repressive epigenetic marks through infection progression. These epigenetic changes are connected to the transcriptional regulation of their expected targets, genes, and transposable elements. Indeed, several genes related to the defense response are targets of both epigenetic marks. Our results highlight another host regulatory mechanism affected by viroid infection, providing further information about the complexity of the multiple layers of interactions between pathogens/viroids and hosts/plants.
Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Heterocromatina , Histonas , Enfermedades de las Plantas , Viroides , Heterocromatina/metabolismo , Heterocromatina/genética , Viroides/genética , Viroides/fisiología , Viroides/patogenicidad , Histonas/metabolismo , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Cucumis sativus/virología , Cucumis sativus/genética , Virus de Plantas/fisiología , Virus de Plantas/patogenicidad , Elementos Transponibles de ADN/genética , Interacciones Huésped-Patógeno/genéticaRESUMEN
Heterochromatin is critical for maintaining genome stability, especially in flowering plants, where it relies on a feedback loop involving the H3K9 methyltransferase, KRYPTONITE (KYP), and the DNA methyltransferase CHROMOMETHYLASE3 (CMT3). The H3K9 demethylase INCREASED IN BONSAI METHYLATION 1 (IBM1) counteracts the detrimental consequences of KYP-CMT3 activity in transcribed genes. IBM1 expression in Arabidopsis is uniquely regulated by methylation of the 7th intron, allowing it to monitor global H3K9me2 levels. We show the methylated intron is prevalent across flowering plants and its underlying sequence exhibits dynamic evolution. We also find extensive genetic and expression variations in KYP, CMT3, and IBM1 across flowering plants. We identify Arabidopsis accessions resembling weak ibm1 mutants and Brassicaceae species with reduced IBM1 expression or deletions. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity and loss of gene body DNA methylation, as cmt3 mutants in A. thaliana mitigate the deleterious effects of IBM1.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Metilación de ADN , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Heterocromatina , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilación de ADN/genética , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Intrones/genética , Histonas/metabolismo , Histonas/genética , Mutación , ADN-Citosina Metilasas/metabolismo , ADN-Citosina Metilasas/genética , Inestabilidad GenómicaRESUMEN
Repression of facultative heterochromatin is essential for developmental processes in numerous organisms. Methylation of histone H3 lysine 27 (H3K27) by Polycomb repressive complex 2 is a prominent feature of facultative heterochromatin in both fungi and higher eukaryotes. Although this methylation is frequently associated with silencing, the detailed mechanism of repression remains incompletely understood. We utilized a forward genetics approach to identify genes required to maintain silencing at facultative heterochromatin genes in Neurospora crassa and identified three previously uncharacterized genes that are important for silencing: sds3 (NCU01599), rlp1 (RPD3L protein 1; NCU09007), and rlp2 (RPD3L protein 2; NCU02898). We found that SDS3, RLP1, and RLP2 associate with N. crassa homologs of the Saccharomyces cerevisiae Rpd3L complex and are required for repression of a subset of H3K27-methylated genes. Deletion of these genes does not lead to loss of H3K27 methylation but increases acetylation of histone H3 lysine 14 at up-regulated genes, suggesting that RPD3L-driven deacetylation is a factor required for silencing of facultative heterochromatin in N. crassa, and perhaps in other organisms.
Asunto(s)
Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Heterocromatina , Histonas , Neurospora crassa , Neurospora crassa/genética , Neurospora crassa/metabolismo , Heterocromatina/metabolismo , Heterocromatina/genética , Histonas/metabolismo , Histonas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Acetilación , Silenciador del Gen , Metilación , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genéticaRESUMEN
The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.
Asunto(s)
Sistemas CRISPR-Cas , Cromatina , Epigénesis Genética , Edición Génica , Inhibidores de Histona Desacetilasas , Humanos , Edición Génica/métodos , Epigénesis Genética/efectos de los fármacos , Cromatina/metabolismo , Cromatina/genética , Inhibidores de Histona Desacetilasas/farmacología , Reparación del ADN , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Línea Celular , Histonas/metabolismo , Eucromatina/genética , Roturas del ADN de Doble Cadena/efectos de los fármacosRESUMEN
Expression of Protocadherin (Pcdh) genes is critical to the generation of neuron identity and wiring of the nervous system. Pcdhα genes are arranged in clusters and exhibit a range of expression profiles, from stochastic to deterministic. Because Pcdhα promoters have high sequence identity and share distal enhancers, how distinct neurons choose which gene to express remains unclear. We show that the interplay between multiple enhancers, epigenetics, and genome folding orchestrates differential readouts of the locus across neurons. The probability of Pcdhα promoter choice depends on enhancer/promoter encounters catalyzed by cohesin, whose extrusion trajectories determine the likelihood that an individual promoter can "escape" heterochromatin-mediated silencing. We propose that tunable locus-specific regulatory elements and cell type-specific cohesin activity underlie the generation of cellular diversity by Pcdh genes.