microRNA-Induced Epigenetic Remodeling during Neuronal Reprogramming of Human Fibroblasts

The ability to generate human neurons that reflect the age of adult individuals will provide a robust cellular platform to model adult-onset neurological disorders using patient-derived neurons. Recent advances in cell fate reprogramming technologies have demonstrated the utility of neuron-enriched genetic factors to convert (reprogram) non-neuronal somatic cells such as dermal fibroblasts directly into neurons. During mammalian neural development, brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124) contribute to the establishment of a neuronal identity by downregulating genes that repress neuronal gene expression. We found that miR-9/9*-124 were capable of efficiently converting human fibroblasts directly to neurons when ectopically expressed, demonstrating the miRNAs’ function as potent cell fate regulators even in the non-neural context. This direct neuronal conversion bypasses the pluripotent/multipotent stem cell stages, thus generating neurons that retain the age of fibroblast donors. Our recent investigation into the mechanism by which miR-9/9*-124 overcomes the cell-fate barrier presented in human fibroblasts leading to the adoption of a neuronal state indicates that extensive changes in the transcriptome and chromatin landscape underlie the miR-9/9*-124-mediated neuronal reprogramming. Time series analyses of transcriptome changes in response to miR-9/9*-124 reveal alterations in genetic pathways during early time points, leading to a sustained and stable transcriptomic switch with an activation of a neuronal program with the simultaneous inactivation of genes related to fibroblast fate. Importantly, miR-9/9*-124 stimulate increased chromatin accessibilities for genomic loci associated with neuronal genes and closing of loci for genes related to fibroblast fate, all coinciding with the adoption of a neuronal identity. Genome-wide assessment of DNA-methylation patterns reveals an emergence of differentially methylation regions that concur as cells adopt a stable neuronal conversion. Our results collectively demonstrate the potency of miR-9/9*-124 as potent chromatin effectors, leading to the reconfiguration of the chromatin state associated with the newly acquired neuronal fate. Furthermore, we find that miR-9/9*-124 also result in the opening of chromatin regions associated with genes expressed in specific subtypes of neurons, yet without the simultaneous gene activation. Thus, the miRNA-induced neuronal state likely allows a cellular state permissive to further specification into distinct neuronal subtypes. By applying additional brain-region specific transcription factors, the miR-9/9*-124-induced neuronal conversion can be instructed to specific neuronal subtypes including cortical neurons, striatal medium spiny neurons, and spinal cord motor neurons, clinically relevant neuronal subtypes affected in many forms of late-onset neurological disorders. Further, we find that generating medium spiny neurons, the primary cell type affected in Huntington’s disease (HD), from HD patients provides a cellular model displaying cellular phenotypes associated with the HD pathogenesis. Together, our study demonstrates the mechanistic insight into how miRNAs generate a neuronal state that can be further guided to distinct neuronal subtype, offering a cellular platform to model adult-onset neurological disorders.