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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.

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Rest regulatory networks

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

Histone K4 Demethylase

HDAC

HDAC

MeCP2

SCP

mSin3

CoREST

DNMT1

REST

HMT K9

HP1

MeCP2

RE1

mCpG

Methyl H3K9

mK9

mK9

mK9

mK9

mK9

mK9

mK9

mK9

mK9

m

m

m

m

m

m

m

m

m

m

Differentiated non-neuronal cells

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 neural-specific 10

Synaptosomal-associated 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

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

d0

d5

d7

d9

d11

d0

d5

d7

d9

d11

Neural differentiation of P19 mouse embryonic carcinoma cells

RA induction

Western blots

Northern blots

REST

miR-9

msin3A

SCP-1

miR-124a

CoREST

U6

Neuronal marker TUJ1


Neural-specific microRNAs likely target com-ponents of the REST co-repressor complex

mCpG

Methyl H3K9

mK9

mK9

mK9

mK9

mK9

mK9

mK9

mK9

mK9

m

m

m

m

m

m

m

m

m

m

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

Plasmid

transfected:

Plasmid

transfected:

mir324

mir124

empty

GFP

empty

mir-9

mir1

msin3A

SCP-1

Loading

control

-actin

Histone K4 Demethylase

HDAC

HDAC

MeCP2

SCP

mSin3

CoREST

DNMT1

REST

HMT K9

HP1

MeCP2

RE1


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 3’UTR

SCP-1 sensor

ADAR1 3’UTR

Control sensor

Control sensor

SCP1 sensor + mir124



A model for REST regulation to enhance neurogenesis

Neurons

Neuronal progenitors

REST high

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

REST-regulated protein coding genes ON

Supported by Crick-Jacobs Center, NSF

Acknowledgements: Eunice Meija, Xinwei Cao


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