RESUMO
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.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Retroelementos , Animais , Sítios de Ligação , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Células-Tronco Embrionárias/citologia , Camundongos , Ligação Proteica , Domínios ProteicosRESUMO
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.
Assuntos
Alelos , Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Nucleossomos , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodosRESUMO
The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilação , Mutação , Precursores de Proteínas/metabolismo , Interferência de RNA/fisiologia , Transgenes/genéticaRESUMO
Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription.
Assuntos
Estruturas Cromossômicas , RNA não Traduzido/genética , Cromossomo X/química , Animais , Feminino , Genes Ligados ao Cromossomo X/genética , Camundongos , RNA Longo não Codificante , RNA não Traduzido/metabolismoRESUMO
In recent years, the Piwi pathway has been shown to regulate the silencing of mobile genetic elements. However, we know little about how Piwi pathways impose silencing and even less about trans-generational stability of Piwi-induced silencing. We demonstrate that the Caenorhabditis elegans Piwi protein PRG-1 can initiate an extremely stable form of gene silencing on a transgenic, single-copy target. This type of silencing is faithfully maintained over tens of generations in the absence of a functional Piwi pathway. Interestingly, RNAi can also trigger permanent gene silencing of a single-copy transgene and the phenomenon will be collectively referred to as RNA-induced epigenetic silencing (RNAe). RNAe can act in trans and is dependent on endogenous RNAi factors. The involvement of factors known to act in nuclear RNAi and the fact that RNAe is accompanied by repressive chromatin marks indicate that RNAe includes a transcriptional silencing component. Our results demonstrate that, at least in C. elegans, the Piwi pathway can impose a state of gene silencing that borders on 'permanently silent'. Such a property may be more widely conserved among Piwi pathways in different animals.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/fisiologia , Inativação Gênica , Animais , Animais Geneticamente Modificados , Perfilação da Expressão Gênica , Modelos BiológicosRESUMO
Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain-sDMA interactions, piRNAs and piRNA targets.
Assuntos
Chaperonas Moleculares/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Elementos de DNA Transponíveis/genética , Feminino , Substâncias Macromoleculares , Masculino , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/metabolismo , Mapeamento de Interação de Proteínas , Interferência de RNA , Proteínas de Ligação a RNA/química , Frações Subcelulares/metabolismo , Testículo/metabolismo , Transcrição Gênica , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
Transposable elements (TEs) are mobile genetic elements that can have many deleterious effects on the fitness of their host. The germline-specific PIWI pathway guards the genome against TEs, deriving its specificity from sequence complementarity between PIWI-bound small RNAs (piRNAs) and the TEs. The piRNAs are derived from so-called piRNA clusters. Recent studies have demonstrated that the piRNA repertoire can be adjusted to accommodate recent TE invasions by capturing invading TEs in piRNA loci. Thus far, no information concerning piRNA divergence is available from vertebrates. We present piRNA analyses of two relatively divergent zebrafish strains. We find that significant differences in the piRNA populations have accumulated, most notably among active class I TEs. This divergence can be split into differences in piRNA abundance per element and differences in sense/antisense polarity ratios. In crosses between animals of the different strains, many of these differences are resolved in the progeny. However, some differences remain, often leaning to the maternally contributed piRNA population. These differences can be detected at least two generations later. Our data illustrate, for the first time, the fluidity of piRNA populations in vertebrates and how the established diversity is transmitted to future generations.
Assuntos
Epigênese Genética , RNA Interferente Pequeno/metabolismo , Peixe-Zebra/genética , Zigoto/metabolismo , Animais , Cruzamentos Genéticos , Elementos de DNA Transponíveis , Feminino , Masculino , RNA Interferente Pequeno/química , Peixe-Zebra/metabolismoRESUMO
RNA interference (RNAi)-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans , Células Germinativas/metabolismo , Proteínas do Tecido Nervoso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Metilação , Mutação , Proteínas do Tecido Nervoso/metabolismo , Estabilidade de RNA/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de SinaisRESUMO
Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2'-O-methyl modification at their 3' end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3'-5' exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing.
Assuntos
Metiltransferases/metabolismo , Oócitos/citologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Uridina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Imunoprecipitação , Hibridização In Situ , Masculino , Metiltransferases/fisiologia , Dados de Sequência Molecular , Mutação/genética , Oócitos/metabolismo , Processamento de Terminações 3' de RNA/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Retroelementos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Testículo/citologia , Testículo/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
The spatial organization of chromosomes is critical in establishing gene expression programs. We generated in situ Hi-C maps throughout zebrafish development to gain insight into higher-order chromatin organization and dynamics. Zebrafish chromosomes segregate in active and inactive chromatin (A/B compartments), which are further organized into topologically associating domains (TADs). Zebrafish A/B compartments and TADs have genomic features similar to those of their mammalian counterparts, including evolutionary conservation and enrichment of CTCF binding sites at TAD borders. At the earliest time point, when there is no zygotic transcription, the genome is highly structured. After zygotic genome activation (ZGA), the genome loses structural features, which are re-established throughout early development. Despite the absence of structural features, we see clustering of super-enhancers in the 3D genome. Our results provide insight into vertebrate genome organization and demonstrate that the developing zebrafish embryo is a powerful model system to study the dynamics of nuclear organization.
Assuntos
Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Elementos Facilitadores Genéticos/genética , Epigenômica , Genoma , Código das HistonasRESUMO
Phase separation represents an important form of subcellular compartmentalization. However, relatively little is known about how the formation or disassembly of such compartments is regulated. In zebrafish, the Balbiani body (Bb) and the germ plasm (Gp) are intimately linked phase-separated structures essential for germ cell specification and home to many germ cell-specific mRNAs and proteins. Throughout development, these structures occur as a single large aggregate (Bb), which disperses throughout oogenesis and upon fertilization accumulates again into relatively large assemblies (Gp). Formation of the Bb requires Bucky ball (Buc), a protein with prion-like properties. We found that the multi-tudor domain-containing protein Tdrd6a interacts with Buc, affecting its mobility and aggregation properties. Importantly, lack of this regulatory interaction leads to significant defects in germ cell development. Our work presents insights into how prion-like protein aggregations can be regulated and highlights the biological relevance of such regulatory events.
Assuntos
Células Germinativas/metabolismo , Oócitos/metabolismo , Oogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Citoplasma/metabolismo , Organelas/metabolismo , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
BACKGROUND: Enhancers, not promoters, are the most dynamic in their DNA methylation status throughout development and differentiation. Generally speaking, enhancers that are primed to or actually drive gene expression are characterized by relatively low levels of DNA methylation (hypo-methylation), while inactive enhancers display hyper-methylation of the underlying DNA. The direct functional significance of the DNA methylation state of enhancers is, however, unclear for most loci. RESULTS: In contrast to conventional epigenetic interactions at enhancers, we find that DNA methylation status and enhancer activity during early zebrafish development display very unusual correlation characteristics: hypo-methylation is a unique feature of primed enhancers whereas active enhancers are generally hyper-methylated. The hypo-methylated enhancers that we identify (hypo-enhancers) are enriched close to important transcription factors that act later in development. Interestingly, hypo-enhancers are de-methylated shortly before the midblastula transition and reside in a unique epigenetic environment. Finally, we demonstrate that hypo-enhancers do become active at later developmental stages and that they are physically associated with the transcriptional start site of target genes, irrespective of target gene activity. CONCLUSIONS: We demonstrate that early development in zebrafish embodies a time window characterized by non-canonical DNA methylation-enhancer relationships, including global DNA hypo-methylation of inactive enhancers and DNA hyper-methylation of active enhancers.
Assuntos
Metilação de DNA/genética , Elementos Facilitadores Genéticos , Epigênese Genética , Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Sítio de Iniciação de Transcrição , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
RNAi is essential for pericentromeric heterochromatic formation in S. pombe, and although Dcr1, the initiator protein of this process, has been biochemically well described, its subcellular localization has remained elusive. In this issue of Developmental Cell, Emmerth et al. now show that Dcr1 is dynamically shuttling between nucleus and cytoplasm, adding new insight into the subcellular mechanics of RNAi.