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

REST

mSin3MeCP2 HDAC SCP

CoREST

HDAC

MeCP2

Histone K4 Demethylase

HMT K9

HP1

DNMT1

RE1mK9

mK9

mK9

mK9 mK9

mK9

mK9

mK9

m

m m m m

m

mm m

m mCpG

mK9 Methyl H3K9

Slide courtesy of XC

REST binds to a ~20 bp sequenceREST levels decrease during neuronal differentiation

REST sites are highly-conserved across evolution

Brain-derived neurotrophic factor

Superiorcervical ganglion neural-specific 10

Synaptosomal-associated protein 25

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

Anti-REST chromatin immunoprecipitation/qPCR for candidate REST sites in P19 cells upon neural induction

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

miR-9-3

miR-29a/29b-1

30e/30c-1 miR135b miR153-1

miR182/96/183miR192/194-2miR219-1

% of chromatin immunoprecipitated

Undifferentiated

Differentiated

Neural-specific microRNAs and REST co-repressors during mouse stem cell

neuronal differentiation

d7d5 d9 d11d0

Neuronal marker TUJ1

REST

msin3A

SCP-1

CoREST

Neural differentiation of P19 mouse embryonic carcinoma cells

d7d5 d9 d11d0

miR-9

miR-124a

U6

Northern blots Western blots

RA induction

Neural-specific microRNAs likely target com-

ponents of the REST co-repressor complex

Plasmid transfected: GFP empty mir-9

-actin

mir1

24

mir1

mir3

24

empty

msin3A SCP-1

Loadingcontrol

Plasmid transfected:

mir-9 and mir-124a downregulate mSin3a and SCP-1, respectively

REST

mSin3MeCP2 HDAC SCP

CoREST

HDAC

MeCP2

Histone K4 Demethylase

HMT K9

HP1

DNMT1

RE1mK9

mK9

mK9

mK9 mK9

mK9

mK9

mK9

m

m m m m

m

mm m

m mCpG

mK9 Methyl H3K9

Neural-specific microRNAs likely target com-

ponents of the REST corepressor complex A sensor assay confirms predicted target sites for miR-124a in the

3’UTR of SCP-1

SCP-1 sensor

Control sensor

SCP-1 3’UTR

ADAR1 3’UTR

Control sensor SCP1 sensor + mir124

Enhancing neurogenesis mir-124 induces neuronal gene expression

in P19 mouse EC cells

0

1

2

3

4

5

6

GAP43 NFH Glur2 ND1

Promoter driving luciferase

Fold change

(mir124/control)

24 hours

48 hours

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