Rest Stability Control By EZH2 Underlies Chromatin Remodeler Switching during Microrna-Mediated Neuronal Reprogramming of Human Adult Fibroblasts

Neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), have been shown to convert human adult fibroblasts to neurons when ectopically expressed through inducing epigenetic changes^1-5. Yet, the mechanism by which these miRNAs, as negative regulators of their downstream target genes, lead to the activation of the neuronal program remains to be defined. Here, we reveal that miR-9/9*-124 evoke a cascade of protein stability network orchestrating the destabilization of REST^{6, 7}, a transcriptional repressor of neuronal genes during neuronal reprogramming of human adult fibroblasts. As such, we identified a function of EZH2^{8, 9}, a lysine-methyl-transferase that stabilized REST by methylating the lysine residue of REST in human adult fibroblasts. Interestingly, REST methylation by EZH2 occurred independently of EZH2’s canonical role as a subunit of Polycomb Repressive Complex 2 (PRC2)^{8, 9}. During neuronal reprogramming, miR-9/9*-124 led to a concurrent repression of EZH2 and REST and an important output of the downregulation of the EZH2-REST axis was the transcriptional activation of BAF53b^10-12, a neuron-specific component of BAF chromatin remodeling complex essential for proper neuronal differentiation and function. Consistent with this finding, extending the expression of EZH2 during neuronal differentiation of human neural stem cells or in the developing mouse brain resulted in the failure to activate BAF53b. These results demonstrate how miRNAs trigger a cascade of protein stability controls to promote the neuronal program and the assembly of neuron-specific BAF complex during neuronal reprograming of human fibroblasts.

1. Victor, M.B. et al. Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts. Neuron 84, 311-323 (2014).

2. Richner, M., Victor, M.B., Liu, Y., Abernathy, D. & Yoo, A.S. MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons. Nat Protoc 10, 1543-1555 (2015).

3. Huh, C.J. et al. Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts. Elife 5 (2016).

4. Yoo, A.S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228-231 (2011).

5. Abernathy., D.G. et al. MicroRNAs Induce a Permissive Chromatin Environment That Enables Neuronal Subtype-specific Reprogramming of Adult Human Fibroblasts. Cell Stem Cell (in press) (2017).

6. Chong, J.A. et al. REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80, 949-957 (1995).

7. Schoenherr, C.J. & Anderson, D.J. The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267, 1360-1363 (1995).

8. Cao, R. et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039-1043 (2002).

9. Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343-349 (2011).

10. Yoo, A.S., Staahl, B.T., Chen, L. & Crabtree, G.R. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460, 642-646 (2009).

11. Yoo, A.S. & Crabtree, G.R. ATP-dependent chromatin remodeling in neural development. Curr Opin Neurobiol 19, 120-126 (2009).

12. Yoo, M. et al. BAF53b, a Neuron-Specific Nucleosome Remodeling Factor, Is Induced after Learning and Facilitates Long-Term Memory Consolidation. J Neurosci 37, 3686-3697 (2017).