1. REST regulatory networks Gene Yeo 1,2 Stefan Aigner 2, Eric Van Nostrand 1,2, Fred H. Gage 2 1 Crick-Jacobs Center of Theoretical and Computational Biology 2 Laboratory of Genetics The Salk Institute for Biological Studies, La Jolla, CA

2. REST repressor/silencing complex silences neuronal genes in non-neuronal cells Differentiated non-neuronal cells REST mSin3 MeCP2 HDAC SCP CoREST HDAC MeCP2 Histone K4 Demethylase HMT K9 HP1 DNMT1 RE1 mK9 m mmmm m mmm m mCpG mK9 Methyl H3K9 Slide courtesy of XC REST binds to a ~20 bp sequence REST levels decrease during neuronal differentiation

3. REST sites are highly-conserved across evolution Brain-derived neurotrophic factor Superiorcervical ganglion neural- specific 10 Synaptosomal- associated protein 25

4. Algorithm to identify conserved REST sites Converged motif

5. Several REST sites are proximal to microRNAs

6. REST-regulated microRNAs are neuronal

7. REST binds in vivo to predicted sites

8. REST occupancy near microRNA genes decreases during neural differentiation Anti-REST chromatin immunoprecipitation/qPCR for candidate REST sites in P19 cells upon neural induction

9. Neural-specific microRNAs and REST co-repressors during mouse stem cell neuronal differentiation d7d5d9d11d0 Neuronal marker TUJ1 REST msin3A SCP-1 CoREST Neural differentiation of P19 mouse embryonic carcinoma cells d7d5d9d11d0 miR-9 miR-124a U6 Northern blots Western blots RA induction

10. Neural-specific microRNAs likely target com- ponents of the REST co-repressor complex Plasmid transfected: GFPemptymir-9 -actin mir124 mir1 mir324 empty msin3ASCP-1 Loading control Plasmid transfected: mir-9 and mir-124a downregulate mSin3a and SCP-1, respectively REST mSin3 MeCP2 HDAC SCP CoREST HDAC MeCP2 Histone K4 Demethylase HMT K9 HP1 DNMT1 RE1 mK9 m mmmm m mmm m mCpG mK9 Methyl H3K9

11. Neural-specific microRNAs likely target com- ponents of the REST corepressor complex A sensor assay confirms predicted target sites for miR-124a in the 3UTR of SCP-1 SCP-1 sensor Control sensor SCP-1 3UTR ADAR1 3UTR Control sensorSCP1 sensor + mir124

12. Enhancing neurogenesis

13. REST high Neuronal progenitors Neurons REST-regulated microRNAs OFF REST-regulated microRNAs ON REST-regulated microRNAs downregulate components of the REST repressor complex REST-regulated protein coding genes ON REST-regulated protein coding genes OFF A model for REST regulation to enhance neurogenesis REST low Acknowledgements: Eunice Meija, Xinwei Cao Supported by Crick-Jacobs Center, NSF

14. REST binds in vitro and in vivo to sites predicted near microRNA genes

15. Characteristics of REST protein-coding targets Conserved REST sites are more enriched near transcriptional start sites