RESUMO
A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.
Assuntos
Cromossomos Humanos X/química , Epigênese Genética , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Inativação do Cromossomo X , Alelos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Cromossomos Humanos X/metabolismo , Ilhas de CpG , Edição de Genes , Inativação Gênica , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes.