Author: Matthew J. McCoy
Publisher:
Published: 2018
Total Pages: 124
ISBN-13:
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Cell fate reprogramming is transforming our understanding of the establishment and maintenance of cellular identity. In addition, reprogramming holds great promise to model diseases affecting cell types that are prohibitively difficult to study, such as human neurons. Overexpression of the brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124) results in reprogramming human somatic cells into neurons and has recently been used to generate specific neuronal subtypes affected in neurodegenerative disorders. However, the mechanisms governing the ability of miR-9/9*-124 to generate alternative subtypes of neurons remained unknown. In this thesis, I report that overexpressing miR-9/9*-124 triggers reconfiguration of chromatin accessibility, DNA methylation, and mRNA expression to induce a default neuronal state. MiR-9/9*-124-induced neurons (miNs) are functionally excitable and are uncommitted towards specific subtypes yet possess open chromatin at neuronal subtype-specific loci, suggesting such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show ISL1 and LHX3 selectively drive conversion to a highly homogenous population of human spinal cord motor neurons. This work shows that modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, thereby providing a general platform for high-efficiency generation of distinct subtypes of human neurons. Since many neurodegenerative diseases occur after development, modeling them requires reprogramming methods capable of generating functionally mature neurons. However, few robust molecular hallmarks existed to identify such neurons, or to compare efficiencies between reprogramming methods. Recent studies demonstrated that active long genes (>100 kb from transcription start to end) are highly enriched in neurons, which provided an opportunity to identify neurons based on the expression of these long genes. We therefore worked to develop an R package, LONGO, to analyze gene expression based on gene length. We developed a systematic analysis of long gene expression (LGE) in RNA-seq or microarray data to enable validation of neuronal identity at the single-cell and population levels. By combining this conceptual advancement and statistical tool in a user-friendly and interactive software package, we intended to encourage and simplify further investigation into LGE, particularly as it applies to validating and improving neuronal differentiation and reprogramming methodologies. Using this tool, I found by single-cell RNA sequencing that microRNA-mediated neuronal reprogramming of human adult fibroblasts yields a homogenous population of mature neurons, and that LGE distinguishes mature from immature neurons. I found that LGE correlates with expression of neuronal subunits of the Swi/Snf-like (BAF) chromatin remodeling complex, such as ACTL6B/BAF53b. Finally, I found that the loss of a functional neuronal BAF complex, as well as chemical inhibition of topoisomerase I, decreases LGE and reduces spontaneous electrical activity. Together, these results provide mechanistic insights into microRNA-mediated neuronal reprogramming, and demonstrate a transcriptomic feature of functionally mature neurons.
