1. “RESCUE OF RE1/NRSE PATHOLOGY IN HUNTINGTON DISEASE” and
“REGULATION OF THE STEM CELL EPIGENOME BY REST”
Chiara Soldati, Angela Bithell, Noel Buckley 1

2. REST (RE1 Silencing Transcription factor ) NSRF (Neuron restrictive silencing factor)
First identified as a regulator of neuronal
genes in non-neuronal cells
Act as repressor of gene transcription
exerts its function by binding to RE1-sites
binds to over 2400 regulatory sites
REST mice die around E10
REST/NRSF
RE1/NRSE
RE1 position-specific scoring matrices
(PSSM)(Johnson, 2006)

3. REST and Stem cell
Rest is not required for the maintenance of stem cell pluripotency (Yamada)
REST is a component of the pluripotency network that includes Oct4,Sox2, a Nanog, which together control differentiation and pluripotency in ESC. (Johnson and Bukley)
REST ablation causes delayed repression of pluripotent genes expression (Oct4,Sox2, a Nanog) during early differetiation (Yamada)
In ESC REST bind and regulate several genes linked to neuronal function (neurotransmitter receptor subunits, neuronal adhesion-associated molecules, synaptic vesicle biology) (Johnson and Buckley)

4. Epigenetics Modifications
A different layer of trascriptional regulation
Influence gene expression
Includes histone modifications and DNA methylation
Chromatin
Heterochromatin and Euchromatin
Nucleosome Basic unit = 147pb wrapped
around Histone octamer 2X H2A H2B H3 H4
Histone C-terminal domain, N-terminal tail
DNA wrapped around histones

5. Histone modification regulates accessibility to gene promoter regions
Acetylation and Methylation regulate accessibility to promoter regions of genes
The developmental programme of embryogenesis is controlled by both genetic and epigenetic Mechanisms
Regulation of chromatin structures is crucial for genome reprogramming during early embryogenesis and for tissue-specific gene expression and global gene silencing.
Regulate the accesso al promotore
Transcriptionally active chromatin
Transcriptionally repressive chromatin

6. REST as epigenetic regulator
BRG1
SMCX
LSD1
co-REST
msin3a
HDAC1/2
RE1/NRSE
REST/NRSF
recruits various histone-modifying and chromatin-remodelling complexes
“RE1 Silencing Transcription Factor Maintains a Repressive Chromatin Environment in Embryonic Hippocampal Neural Stem Cells” Greenway and Buckley)
Low level of H3K9ac and H4ac
Chromatin
Silencing of REST in NSC produces changes in the local epigenetic signature of several target genes

7. AIM Investigate the role of REST in genome and epigenome
regulation of ESC
Investigate the role of REST in neural precursor generation and in neuronal differentiation
Investigate the role of rest in cell maintaining and differentiation at the epigenetic level and trascriptional level

8. Cre/loxP-Mediated REST Inactivation
Flped allele
Exon 2
loxP
loxP
FloRES (D4, D2, C8)
recombination of DNA
between loxP site
Cre
Sox2
DAPI
Oct3/4
DAPI
Exon 2
The Cre protein is a site-specific DNA recombinase, that catalyse the recombination of DNA between specific sites on DNA.These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence where recombination can occur
FloREScre(C18,C23,C11,C28)
Knockout allele
loxP
Exon 2
Sox2
DAPI
Oct3/4
DAPI

9. REST protein by western blot with 2 different antibody: Santa Cruz (internal) Upstate (C-terminal)
D4,C8= floRES C18, C23, C28 =floREScre

10. REST GENE by PCR on DNA

11. REST and transcription in Stem Cell
REST and epigenome in Stem Cell
REST and neural precursors Generetion

12. Real Time PCR gene regulated and not regulated by REST
FloRES D4
FloREScre C18
Unc13a encodes a synaptic vesicle protein
Snap25, a regulator of neural transmitter release [20], and Cels3r, a Gprotein-
coupled receptor,
“REST Regulates Distinct
Transcriptional Networks in
Embryonic and Neural Stem
Cells” Rory Johnson
Celsr13 Golga7b Unc13a Chga Snap25 Nps4a Vrk3 REST

13. TRANSCRIPTOME ANALYSIS FloRES D4 VS FloREScre C18
RNA sequencing
All transcript mRNA (non-coding RNA, small RNAs)
Trascriptional structure (5’and 3’ end, splicing, and other post trascriptional modification) relative level of expression
Novel trascribed reagions
Microarray
measure changes in expression levels
next-generation sequencing
RNA sequencing
All species of transcript mRNA (non-coding RNA, small RNAs)
Trascriptional structure of gene (5’and 3’ end, splicing, and other post trascriptional modification)
Structure/level of expression
Novel trascribed reagions

14. REST and transcription in Stem Cell
REST and epigenome in Stem Cell
REST and neural precursors Generetion

15. Epigenome investigation by Chromatin Immunoprecipitatin assay (ChIP)
H3K9acetylated
H4 acetylated
Investigate the structure of chromatin around RE1

16. ChIP with H3K9ac an H4ac antibody
FloRES D4
FloREScre C18
Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk Chga
Ctrl
Removal of REST not sufficient for target gene derepression (gene activation can requires activating transcription factors)
REST KO
Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk Chga

17. ChIP with REST antibody
REST IgG
FloRES D4
FloREScre C18
Snap25 Golga7b Celsr Unc13a Npas Vrk Chga
REST can affect histons modification independently from gene transcription
i.e. Vrk3 and Npas4: gene expresion is not regulated by REST (in STEM CELL) But
H3K9 and H4 acetylation change around RE1
Genome wide analysis by ChIP-seq

18. REST and transcription in Stem Cell
REST and epigenome in Stem Cell
REST and neural precursors Generation

19. “Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell”
Conti and Smith 2005
EGF
bFGF
EGF
bFGF
EGF
bFGF
(P0)
(nP)
N2B27 ES
Rosettes
aggregates RG
NSC
Nestin
Sox1
LIF
Nestin
Vimentin
RC2
BLBP

20. 99% Nestin+ 80-90% Ki67+ -Radial Glia marker FloRES (P0) FloRES (P7)
RC2 olig2
Dapi
Blbp
Dapi
60-70%
Radial Glia like cells
Vimentin Pax6
Dapi
Sox2
Dapi
GFAP
bIIItub
Dapi
100%
SOX2 +

21. REST KO affect NSC maintaining
FloREScre (P0)
REST KO affect NSC maintaining
By accelerate the switch between Neuroepithelial like to Radial Glia like cells
By increase spontaneous differentiation
99% Nestin+
70-80% Ki67+
Nestin
Ki67
FloREScre (P1)
Vimentin Pax6
Dapi
RC2 olig2
Dapi
Blbp
Dapi
GFAP
bIIItub
Dapi
Sox2
Dapi
GFAP
bIIItub
Dapi
GFAP
bIIItub
Dapi

22. To summarize REST is a epigenetic regulator in Stem Cell
It doesn't affect neural precursors generation
It affect neural precursors maintaining

23. Work in progress Epigenome Differentiation Transcriptome
Analyze ChIP seq data
HDAC1/2 ChIP and
His tone deacetylase inhibitor
Transcriptome
Analyze Microarray and RNA seq data
Differentiation
Expression of pluripotency and differentional marker from ESC to RGC (Oct3/4, Nanog, Sox2, Gata4, Nestin, Sox1, Pax6, Vimentin, bIIItubulin)
Cloning FloRES and FloREScre NSC to get a population of RGC to investigate late differentiation

24. Thanks to… Noel J Buckley Genome Institute Singapore Angela Bithell
Cass Johnston
Matthew Burney
Alessandro Michelucci
Genome Institute Singapore
Kee-Yew Wong
(microarray) 24