Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs

Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchest...

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Published inCell stem cell Vol. 28; no. 1; pp. 127 - 140.e9
Main Authors Cates, Kitra, McCoy, Matthew J., Kwon, Ji-Sun, Liu, Yangjian, Abernathy, Daniel G., Zhang, Bo, Liu, Shaopeng, Gontarz, Paul, Kim, Woo Kyung, Chen, Shawei, Kong, Wenjun, Ho, Joshua N., Burbach, Kyle F., Gabel, Harrison W., Morris, Samantha A., Yoo, Andrew S.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 07.01.2021
Subjects
Cell Differentiation
cell fate
Cellular Reprogramming - genetics
Chromatin
chromatin regulation
direct conversion
epigenetics
Fibroblasts
Humans
microRNA
MicroRNAs - genetics
neuronal reprogramming
non-coding RNA
single-cell RNA-sequencing
Transcription Factors - genetics
single-cell RNA-sequencing
direct conversion
non-coding RNA
cell fate
chromatin regulation
microRNA
neuronal reprogramming
epigenetics
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Summary:Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9∗-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade. [Display omitted] •miR-9/9∗ and miR-124 synchronously reprogram human adult fibroblasts to neurons•miRNAs induce fibroblast fate network erasure and then neuronal program activation•miRNAs directly target transcription factors KLF4 and KLF5 to erase fibroblast fate•Small nuclear RNA 7SK acts downstream of miRNAs to activate neuronal fate Direct cell conversion generally requires reprogramming effectors to simultaneously induce target cell-type-specific fate programs and suppress starting cell identity. Yoo and colleagues reveal that microRNAs 9/9∗ and 124 direct uniform conversion of human fibroblasts by first erasing fibroblast identity and then inducing transition to a neuronal state.
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Present address: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
Present address: Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115, USA
These authors contributed equally.
D.G.A. performed moto-miN reprogramming. W.K. and S.A.M. prepared scRNA-seq libraries and M.J.M. performed analyses of scRNA-seq data for moto-miNs and gene networks. J.K. and W.K. performed RNA velocity analyses. For miNs and day 10 to 15 moto-miNs, K.C. reprogrammed cells and prepared scRNA-seq libraries, and J.K. performed all analyses of scRNA-seq. K.C. designed and analyzed post-mitotic cells with J.N.H. and K.F.B. and performed fate erasure ATAC-seq and RNA-seq experiments. J.K. and K.C. performed analyses. Y.L. performed AGO-HITS-CLIP and B.Z., S.L., P.G. analyzed the data. Y.L., K.C., and Y.L. performed KLF experiments. K.C. performed 7SK knockdown experiments. A.S.Y., K.C., and W.K.K. designed ATAC-seq and RNA-seq experiments. A.S.Y. and M.J.M. designed LGE experiments with H.W.G.’s inputs. A.S.Y., M.J.M., K.C., J.K., and Y.L. wrote the manuscript. A.S.Y. supervised the project.
Author Contributions
ISSN:1934-5909
1875-9777
DOI:10.1016/j.stem.2020.08.015