RESUMO
Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.
Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Cromatina/metabolismo , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , Técnicas de Transferência Nuclear , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Linhagem Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , Clonagem Molecular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Oócitos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Xenopus laevisRESUMO
Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host's single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Edição de Genes/métodos , Recombinação Homóloga/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Caulobacter crescentus/metabolismo , DNA/química , DNA/genética , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/metabolismo , Recombinação Homóloga/genética , Lactococcus/metabolismo , Mycobacterium smegmatis/metabolismo , Domínios Proteicos/genéticaRESUMO
Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as "genome writing" and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Genoma/genética , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Eucariotos/genética , Edição de Genes/métodos , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.