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REST regulatory networks Gene Yeo1,2 Stefan Aigner 2, Eric Van Nostrand1,2, Fred H. Gage2 1Crick-Jacobs Center of Theoretical and Computational Biology 2Laboratory of Genetics The Salk Institute for Biological Studies, La Jolla, CA REST repressor/silencing complex silences neuronal genes in non-neuronal cells Differentiated non-neuronal cells m mK9 mCpG Methyl H3K9 HDAC MeCP2 Histone K4 Demethylase HDAC SCP mSin3 CoREST REST mK9 m mK9 m mK9 RE1 m mK9 m m HMT K9 m mK9 MeCP2 m m DNMT1 HP1 mK9 mK9 m mK9 REST binds to a ~20 bp sequence REST levels decrease during neuronal differentiation Slide courtesy of XC REST sites are highly-conserved across evolution Superiorcervical ganglion neuralspecific 10 Synaptosomalassociated protein 25 Brain-derived neurotrophic factor Algorithm to identify conserved REST sites Converged motif Several REST sites are proximal to microRNAs REST-regulated microRNAs are neuronal REST binds in vivo to predicted sites REST occupancy near microRNA genes decreases during neural differentiation 0.8 0.7 0.6 Undifferentiated Differentiated 0.5 0.4 0.3 0.2 0.1 iR 21 91 m iR 18 2/ 96 /1 83 m iR 19 2/ 19 42 m iR 13 5b m 30 e/ 30 c1 iR 15 31 m m iR -9 -3 iR -2 9a /2 9b -1 0 m % of chromatin immunoprecipitated Anti-REST chromatin immunoprecipitation/qPCR for candidate REST sites in P19 cells upon neural induction Neural-specific microRNAs and REST co-repressors during mouse stem cell neuronal differentiation Neural differentiation of P19 mouse embryonic carcinoma cells RA induction Western blots Northern blots d0 d5 d7 d9 d11 miR-9 miR-124a d0 d5 d7 d9 d11 REST msin3A SCP-1 CoREST U6 Neuronal marker TUJ1 Neural-specific microRNAs likely target components of the REST co-repressor complex mir-9 and mir-124a downregulate mSin3a and SCP-1, respectively Plasmid transfected: GFP Plasmid transfected: empty mir-9 msin3A SCP-1 Loading control -actin m mK9 mCpG Methyl H3K9 HDAC MeCP2 Histone K4 Demethylase HDAC SCP mSin3 CoREST REST mK9 m mK9 m mK9 RE1 m mK9 m m HMT K9 m mK9 MeCP2 m m DNMT1 HP1 mK9 mK9 m mK9 Neural-specific microRNAs likely target components of the REST corepressor complex A sensor assay confirms predicted target sites for miR-124a in the 3’UTR of SCP-1 SCP-1 3’UTR SCP-1 sensor ADAR1 3’UTR Control sensor Control sensor SCP1 sensor + mir124 Enhancing neurogenesis mir-124 induces neuronal gene expression in P19 mouse EC cells 6 Fold change (mir124/control) 5 4 24 hours 48 hours 3 2 1 0 GAP43 NFH Glur2 Promoter driving luciferase ND1 A model for REST regulation to enhance neurogenesis Neuronal progenitors REST high Neurons REST low REST-regulated microRNAs ON REST-regulated microRNAs downregulate components of the REST repressor complex REST-regulated microRNAs OFF REST-regulated protein coding genes OFF Acknowledgements: Eunice Meija, Xinwei Cao REST-regulated protein coding genes ON Supported by Crick-Jacobs Center, NSF
