“RESCUE OF RE1/NRSE PATHOLOGY IN HUNTINGTON DISEASE”

and

“REGULATION OF THE STEM CELL EPIGENOME BY REST”

Chiara Soldati, Angela Bithell, Noel Buckley

RE1/NRSE

REST/NRSF

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

REST (RE1 Silencing Transcription factor ) NSRF (Neuron restrictive silencing factor)

RE1 position-specific scoring matrices (PSSM)(Johnson, 2006)

binds to over 2400 regulatory sites

REST mice die around E10

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)

Epigenetics Modifications

A different layer of trascriptional regulation

Influence gene expression

Includes histone modifications and DNA methylation

Heterochromatin and Euchromatin

Nucleosome Basic unit = 147pb wrapped

around Histone octamer 2X H2A H2B H3 H4

Histone C-terminal domain, N-terminal tail

Chromatin

Histone modification regulates accessibility to gene promoter regions

Transcriptionally active chromatin

Transcriptionally repressive chromatin

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.

REST as epigenetic regulator 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

BRG1SMCX

LSD1co-RESTmsin3a

HDAC1/2

HDAC1/2

RE1/NRSE

REST/NRSF

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

Exon 2

Flped allele

loxPloxP

Exon 2

Cre

loxP

Exon 2

Cre/loxP-Mediated REST Inactivation

Knockout allele

FloRES (D4, D2, C8)recombination of DNA between loxP site

FloREScre(C18,C23,C11,C28)

Oct3/4DAPI

Sox2DAPI

Oct3/4DAPI

Sox2DAPI

REST protein by western blot with 2 different antibody:

Santa Cruz (internal) Upstate (C-terminal)

RESTREST

D4,C8= floRES C18, C23, C28 =floREScre

UpstateSanta cruz

REST GENE by PCR on DNA

REST and transcription in Stem Cell

REST and epigenome in Stem Cell

REST and neural precursors Generetion

“REST Regulates Distinct Transcriptional Networks in Embryonic and Neural Stem Cells” Rory Johnson

Real Time PCR gene regulated and not regulated by REST

ES RNA expression

floRESD4

floREScre C18

floRESD4

floREScre C18

floRESD4

floREScre C18

floRESD4

floREScre C18

floRESD4

floREScre C18

floRES D4

floREScre C18floRES

D4

floREScre C18

floRES D4 flo

REScre C18

0

50

100

150

200

250

300

350

400

450

500

%vs

flo

RE

S D

4

FloRES D4

FloREScre C18

Celsr13 Golga7b Unc13a Chga Snap25 Nps4a Vrk3 REST

TRANSCRIPTOME ANALYSIS FloRES D4 VS FloREScre C18

Microarray

measure changes in expression levels

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

REST and transcription in Stem Cell

REST and epigenome in Stem Cell

REST and neural precursors Generetion

Epigenome investigation by Chromatin Immunoprecipitatin assay (ChIP)

H3K9acetylated

H4 acetylated

Investigate the structure of chromatin around RE1

FloRES D4

FloREScre C18

ChIP with H3K9ac an H4ac antibody

H3K9 acetyalted

0

5

10

15

20

25

vs f

loR

ES

D4

/ H3

Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk3 Chga

H4 acetyalted

0

1

2

3

4

5

6

vs f

loR

ES

D4 /

H3

Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk3 Chga

Ctrl

REST KO

REST

0

5

10

15

20

25

30

35

ov

er

M4

co

din

g

FloRES D4

FloREScre C18

REST IgG

Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk3 Chga

ChIP with REST antibody

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

REST and transcription in Stem Cell

REST and epigenome in Stem Cell

REST and neural precursors Generation

ES aggregatesRosettes NSCNestinSox1

LIF

N2B27

EGFbFGF

“Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell”Conti and Smith 2005

RGNestinVimentinRC2BLBP

EGFbFGF

EGFbFGF

(P0) (nP)

Nestin

Ki67

FloRES (P0)

FloRES (P7)

99% Nestin+

80-90% Ki67+

-Radial Glia marker

60-70%

Radial Glia like cells

100%

SOX2 +

BlbpDapi

Vimentin Pax6Dapi

RC2 olig2Dapi

Sox2Dapi

GFAPbIIItubDapi

Nestin

Ki67

FloREScre (P0)

BlbpDapi

Vimentin Pax6Dapi

RC2 olig2Dapi

GFAPbIIItubDapi

GFAPbIIItubDapi

GFAPbIIItubDapi

Sox2Dapi

FloREScre (P1)

99% Nestin+

70-80% Ki67+

REST KO affect NSC maintainingBy accelerate the switch between Neuroepithelial like to Radial Glia like cells

By increase spontaneous differentiation

To summarize

REST is a epigenetic regulator in Stem CellIt doesn't affect neural precursors generationIt affect neural precursors maintaining

Work in progress

EpigenomeAnalyze ChIP seq dataHDAC1/2 ChIP and His tone deacetylase inhibitor

TranscriptomeAnalyze Microarray and RNA seq data

DifferentiationExpression 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

Thanks to…

• Noel J Buckley • Angela Bithell • Cass Johnston• Matthew Burney• Alessandro Michelucci

Genome Institute Singapore

• Kee-Yew Wong

(microarray)