Epigenetic enzymes as drugable targets for neurological disease
Christopher WynderPTM Discoveries
Epigenetics:so what• Epigenetics is
responsible for the variation seen in nature amongst close relations
• Epigenetic regulation also provides organisms a mechanism to “tune” gene expression based environment (e.g. low dietary fat and brain development OR long term injury-TBI, stroke)
Epigenetics also explains how dogs have so much variation
Twin studies show that many differences in Autism spectrum disorders seen between siblings is likely epigenetics
Or due to mutations in epigenetic regulators
Epigenetic regulation has multiple jobs in the brain
Stem cell
During development (and regeneration), epigenetics plays a role in what kind of neurons In the brain, Epigenetic
modifications are reused to modify synaptic plasticity
Neurons have very few unique genes defining their sub-typeEpigenetics modifies the levels of specific genes to alter the physical parameters of the neurons.
Epigenetic definition• “Non-genetic events” which result in stable
and inheritable gene expression patterns.• Epigenetic changes are LONG term changes to
shape the portions of the genome that are preferentially expressed
• Particularly relevant during development• Regulation of histone modifications are a
significant and changeable example of an epigenetic modification.
Epigenetic changes are about setting the
context for cell to nucleus signaling.
Limiting the response to any given stimuli
Epigenetics is the genome’s attempt at grammar
• There are approx. 15 000 genes.• Each one has a specific use and time when it should be used.• In Epigenetics, is essentially how genes are organized and how
a cell decides which ones to use at a particular point in time.• Histone modifications give context to allow words to be used
in the correct order.• DNA methylation is used as the bookmarks to define chapters
(e.g. the “make a heart” or “respond to IGF”)
See Jane Stop make Run destroy brain Skinheart jeans workcells
See Jane Stop make Run destroy brain Skinheart jeans workcells
Maintaining expression of genes during differentiation
• Gene regulation is divided into 2 sub-regions, regulatory and promoter, and the transcribed region (gene encoding)
• In general, histone modification of the ORF is a on/off mark due to the absolute requirement of these marks for RNA Polymerase read through rate
• Histone modifications in the promoter generally work by modifying RPol’s access
• Histone modifications in the regulatory region(s) are the “tuner” increasing or decreasing of DNA binding transcription factors to their elements
jmjN Domain - possible adaptor for protein-protein interaction
A/T Rich interaction Domain - DNA binding
PHD Domain (zinc finger similar to RING and FVYE domains)- Chromatin binding a triplicate of PHDs in KDM5s
jmjC histone demethylase - Catalytic domain a-ketoglutaric dehydrogenase
Zn finger Domain - C5H2 zinc finger DNA binding domain (chromatin binding)
KDM5b
KDM5c/d65% Homology
1 1544
1535/1516
KDM5 family structure
Wynder, C; Doughty, M; and Stalker, L. Epigenomics, June 2010
Neuron
During neural differentiation, neurons must establish communication.
Tu parle francais?Do you
speak english?
Yes
Cells use KDM5 family members to tune everything from the receptors and synaptic shuttling machinery to the metabolic machinery.
This comprehensive control allows the neurons to control both short term plasticity and long term “gene memory”
KDM5b expressed several layers
KDM5b expression increased after injury
Recycling of cell cycle genes during neural differentiation
KDM5b & ctargets
Frank and Tsai, Neuron 62 (2009)
Step-wise acclimation of histone modifications regulate neural differentiation
3meH3K4
RPolIII
Low read through rate, low amounts of mRNA made, therefore low amounts protein, allows the cell to block the expression of cell lineage genes without 2nd signal.
Extremely low to no read through.3meH3K27
HDM: KDM5(s)HMT:KMT6
HDM: KDM6(s)HMT:KMT2(s)
KDM5b (aka JARID1b/PLU1)
Adapted from Shilatifard Ann.Biochem 2006
Regulating the regulators
ApoptosisPro-neural
Pro-self renewal
Pro-neural
self renewal
Stage specific:Seq. specific TFs:
Oct4, Sox2, FoxD3 NeuroD2, Sox1, FoxG1 Sox17, NGN2 Sox1, FoxD3
PluripotentStem Cell
Neural Progenitor
DifferentiatedNeuron
NeuralStem Cell
Ubiquitous factorsChromatin/histone Regulators:
KDM5b, Ring6a KDM5b, KDM5c, Ring6a KDM5c, Ring6a KDM5b, Ring6a
How do you control Ubiquitous factors to modulate specific events?
Conserved mechanisms
KDM5
Moshkin et al, Mol. Cell 2010
In Drosophila KDM5Is localized by interaction of the NAP1-PF1 complex with Gro-CtBP
NAP1 is a H2B-H2A dimerBinding proteinThought to be a chaperone protein
In vitro1. Recombinant proteins2. Immunopurified complexes from ESCs (or tissue)
Testing epigenetic mechanisms in stem cells
rKDM5b
Enzyme assaysWB from Nuc. Extracts mESCs
+In vivo1. Harvest spheres for RT-PCR and ChIP2. Functional assay based cell markers(proteins)
Differentiation assays
Stalker L and Wynder C. Chapter 27. Methods in Molecular Biology. 2012
TLE4
H3
H4 H2A
H2BK4 K43
H3
H4 H2A
H2BK4
meme
me
K43me
me
Stem Cell
Cell cycle inhibitor
RERE
PluripotentSelf-renewing, proliferative
KDM5b
PHD domainH3
H4 H2A
H2BK4
meme
me
K43
H3
H4 H2A
H2BK4
meme
me
K43
BHC80
Multipotent Proliferative
e.g. Neural stem cell
Stem Cell
Cell cycle inhibitor
KDM5b
?
KDM5b
Lineage Committed e.g. neuron
H3
H4 H2A
H2BK4
meme
me
K43me
me
Cell cycle inhibitor
BHC80
TLE4
H3
H4 H2A
H2BK4 K43
Stem CellRERE
KDM5b
KDM5b recruitment and activation
mTcf3 is a developmental target
Fold
of T
cf3
tran
scrip
t
**
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Control JARID1b
3meH3K4JARID1b
Fold
of c
hang
e in
m
Tcf3
pro
mot
er
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control KDM5b
**
****
0
0.5
1
1.5
2
2.5
3
RNA 3meH3K4 KDM5b
ControlKDM5b
Fold
of c
hang
e RN
A or
D
NA
bind
ing
Stem Cells:
mTcf3 Nanog Self-renewal
Differentiation
mTcf3 Nanog Cell fate genesTransient transfection
Stable mESCs NeurospheresDey BK., et al, (Wynder C.) 2008 Mol. Cell Biol.
TLE4 expression brain
TLE4(Gro homologue) and nucleosomal demethylation
n=3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
****
n=6
Nuc. r.KDM5b r.TLE4 r.KDM5br.TLE4
Fold
cha
nge
in
Nuc
leos
omal
H3K
4me3
r.KDM5dr.TLE4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
**
n=6
Fo
ld c
ha
ng
e in
N
ucl
eo
som
al 3
me
H3
K4
H3K4me3 t=0 H3K4me3 + KDM5b t= 60 min
H3K4me3 + KDM5b + TLE4 t= 60 min
0102030405060708090
100
% m
ethy
latio
n re
mai
ning
H3
K4MeMeMeKDM5b
MeMeMe K4
H3
X
0
2
4
6
8
10
12
Fo
ld c
ha
ng
e in
N
ucl
eo
som
al 3
me
H3
K4
****
b-actin TLE4 IPCcNSP IP
**n=3
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Fo
ld c
ha
ng
e in
N
ucl
eo
som
al 3
me
H3
K4
b-actin TLE4 IPTLE4 IPmESCCcNSP
**
mESC
** n=3
Q GP CcN SP WD40Repeat
CcN SP
Core regulatory domains
Potential inhibitor
Inhibiting KDM5 function
Transfect stable lines
mESCs (NSPC, iPSCs)
TLE4
2meK43-H2B 3meH3K40
0.2
0.4
0.6
0.8
1
1.2 Controlr.KDM5b n=9*
H2BK43me2 H3K4me3
**
**
HD
M a
ctivi
ty o
n m
ixed
pe
ptide
s
H3K4me2
H3K4me3
H3K9me2
H3K9me3
H2BK43me2
H2BK43me3
H3K23me2
H3K23me3
H3K36me2
H3K36me3
H3K27me2
H3K27me3
H2BK46me2
H2BK46me3
H2BK108me2
H2BK108me3
H4K20me2
H4K20me30
10
20
30
40
50
60
70
80
90
100
KDM5b demethylase activity (60 min)%
subs
trat
e de
met
hyla
tion
Control(known substrates)
0 30 60 90 120 150 180 2100
10
20
30
40
50
60
70
80
90
100
H3K4me3
H2BK43me2
Time [min]
% d
emet
hyla
ted
subs
trat
e
r.KDM5b removes methyl groups from K43 faster than K4
K4
me
H3 H2B
meme
K43
meme
KDM5b
TLE4
TSS
KDM5s are regulated by 2 part system
Wnt/GSK3Notch
Wnt/GSK3Stats/CK2
NGF/Erk2Steroidscatabolism
KDM5b
2meK43H2B
3XflagH2BH2B
3XFlH2BmESC
3XFlK43A-mESC
3XFlH2BNS
3XFlK43A-
NS
Flag M2
K43me2 is enriched in progenitors but not stem cells
Doublecortin2meK43 H2B
Nose tail
Phospho-H32meK43 H2B
TUJ12meK43 H2B
2meK43 H2B Dorsal Root ganglia
3XFL
-H2B
3XFL
-K43
A
20mM
20mM 20mM
20mM
100mM
100mM
Day 10Day 6 Day 10DAPISox1
DAPISox1
hg
kj
Loss of H2BK43 methylation phenocopies KDM5b over-expression.
Linking biochemistry to biological properties-Future directions
1. Define relationship between KDM5 and cell signaling (MS sequencing and verification)
2. Define the cell biology that is altered by modulation of this system (Post injury NSCs and iPSCs [NSCs v skin])
3. What is the role of these proteins in both injury and recovery (mouse models)
APP
ROR
a-CateninKDM5bPARP
Ku70
KDM5bDNA Damage
RERE
Stem CellWnt repression
KDM5b
Proliferation
Notch Signaling
TLE4 TLE4-s
KDM5bADAMST8/12?
Variable of recruiters of KDM5b activity
OR
PRSS23
PHF23
APP
BHC8
0
Par3
K43MeMe
TLE4
Ku70hnRNPA2
D
Ring6a
ROR
Akt
a-Catenin KDM5bPARP
PKC
Neural differentiation
NICDWnt
b-Catenin
Anti YFP(TLE4)
FLAG RERE (FL)
FLAG
-RER
EYf
p
FLAG
-RER
EYf
p KD
M5b
FLAG
-RER
EYf
pTLE
4
FLAG
-RER
Eal
one
Anti KDM5b
IP:FLAG
RERE regulates KDM5b interactions and localization
FLAG
-RER
E
YFP-
KD
M5b
YFP-
TLE4
YFP
C1
Whole cell Extract
Chromatin Pellet
antiKDM5b
antiKDM5b
antiGAPDH
antiH3
Linking biochemistry to biological properties-Future directions
1. Define relationship between KDM5 and cell signaling (MS sequencing and verification)
2. Define the cell biology that is altered by modulation of this system (Post injury NSCs and iPSCs [NSCs v skin])
3. What is the role of these proteins in both injury and recovery (mouse models)
Why iPSCs?
• Stem cells have the ability to differentiate into all cell lineages and self renew• Recent advances have allowed us to convert skin into stem cells• Since Rett syndrome mutations happen in all cells we can take skin and make stem cells by
epigenetically re-programming the skin• iPS can be used as a model system to monitor neural differentiation to test where the errors are
and possibly what effect therapeutics have on these errors
Endoderm
Mesoderm
EctodermNeurons
RBC
Skeletal system
Pancreatic cells
Skin cells
Stem cell
Stomach cell
Harvest spheres for RT-PCR and ChIP
Represents “Day 1” of neurodifferentiation assay
Harvest Day 3
Harvest Day 14
Harvest Day 10
Harvest Day 8Harvest Day 6
Harvest Day 5
Harvest Day 4
Mainly Neural Stem
Completely Differentiated
mESCs or iPSCs
Testing epigenetic mechanisms
1
2
Abrogation of KDM5/Co-factor here to elucidate the role of this complex in acquisition of neural lineage.
Epigenetics of differentiation, can transient expression block/enhance terminal differentiation
(can use adult sphere forming cells including Breast, Prostate from human/mouse)
3Cell lineage selection; can expression during terminal differentiation, modify the type of neuron that is made
ORCause de-differentiation/proliferation (iPSC/Cancer)
K4
me
K4
me
meme
meme
K43
meme
K43
meme
Co-F
KDM5
TLE4
TSS
Defining the epigenetic mechanism of neural specification
1. How does recruiters choose the which KDM5
2. Is the interaction antagonistic
3. How general is this model i.e. how many other recruiters are there?
1. How is TLE4 recruited to KDM5 loci?
2. What is the role of Post-translation modifications in TLE4 localization
Syn Ab/CcNSP construct
*
**
0123456789
10
Day 4
Fo
ld c
ha
ng
e in
nu
mb
er
of
sph
ere
s fo
rme
d
n=3
TLE4 CcNSPControl
**
**
0%
20%
40%
60%
80%
100%
120%
140%
160%
1
Fo
ld c
ha
ng
e in
nu
mb
er
of
sph
ere
s p
late
d
n=3
TLE4 CcNSPControl
TLE4 affects Neural differentiation
TLE4 function is required for neural differentiation
TUJ1CCNSP
Sox1CCNSP
Transfection of the CCNSP Dom-Neg construct blocks both early neural markers(Sox1) and late markers (TUJ1)
b c
feEYFP
EYFP
TLE4 CcNSP
TLE4 CcNSP
mCerulean TLE4 CcNSP
h iSox1EYFP
Sox1TLE4
Sox1CcNSP
20 um
50 um
50 um
a
d
Day 4
Day 7
g
Day 2
Day Four Day Seven0
0.2
0.4
0.6
0.8
1
1.2ControlTLE4CcNSP
NeuroD2 Expression
Fold
Cha
nge
in E
xpre
ssio
nCo
mpa
red
to c
ontr
olj
**** **
Understanding how KDM5 activity is integrated into cell function
C.C. Inhibitors Differentiation Cell lineage
GSK3/Erk BHC80
Ring6a
J1b
KMTx
K43
MeMe
OR
BHC80
TLE4TLE4
Block interactionTest affect during
neural diff.(Glial v Neuron)
Block interactionTest affect during
neural diff.(Glial v Neuron)
No peptide TAT peptide TATH2B37-49(K43me0)
mES
Cs48
hrs
post
Neural diff (3 days) Neural diff (3 days) Neural diff (2 days)
Neu
ral d
iffer
entia
tion
4-5
days
pos
t
18 hrs after passage48 hrs
Change to neural differentiation mediamESC mediaNS NS NS
Day 0 Day 1 Day 3
48 hrs
Inhibitory peptide Gently remove intacted spheres by pipette
Change to ULB plates
Control K43pep Het0
0.5
1
1.5
2
2.5
TCF3
tran
scrip
tion
TCF3
pro
mot
er
* *
0
5
10
15
20
25
30*
Control K43pep
N=6
N=3
Peptide mimics KDM5b+/-
Addition of H2BK43me0 peptides causes the formation of neurospheres within 48 hours
Neu
ral F
ilam
ent
b-T
ubul
in II
I
Phas
e Co
ntra
st
Control K43pep0
0.5
1
1.5
2
2.5
3B-
TubI
I tra
nscr
iptio
n*
N=3
Linking biochemistry to biological properties-Future directions
1. Define relationship between KDM5 and cell signaling (MS sequencing and verification)
2. Define the cell biology that is altered by modulation of this system (Post injury NSCs and iPSCs [NSCs v skin])
3. What is the role of these proteins in both injury and recovery (mouse models)
Epigenetic modifications during programming
• In mESCs H3K4 demethylases can block differentiation when exogenously expressed.
• They act by repressing cell lineage factors.• In non-stem cells (e.g HEK293 or HeLa) they can
activate transcription of stem cell factors (Oct4, Sox2 and Nanog).
This means that altering KDM5 function in cells can coax stem cells towards a preferred lineage.
Oct4 genes Sox2 genes KLF4 genes
KLF4
Taking advantage of signal dependent epigenetic activity
In mESCs addition of Wnt3a mediates recruitment of H3K4 demethylase(es) toKLF4 target genes
Wnt3a
GSK3
J1b??
KDM5b is sufficient for Nanog expression in Skin cells
GFP+ Skin from mice
Transfected with KDM5b
Add mESC media
Culture 10d.
Nanog
Nanog
Differential Repression profiles
0
2
4
6
8
10
12
14
16
J1b-MEFmedia J1b-MEFmedia.3 J1b-mES.3 J1b-mES+Wnt3a.1 J1b-mES+Wnt3a.2 J1b-Neural.1
BRCA1
p27
14-3-3
Colony formation in skin.
• KDM5b induces iPS-like cells.• This induction is dependent on
the extra-cellular factors• In appropriate media cells
become arborized.• KDM5b alone is not sufficient
Putative “iPS clone”
Pre-colony
Neural mediaKDM5b transfected
Therapeutic neural conversions
• KDM5b has the modulate the expression of a variety of its target genes.
• The upstream signaling defines which targets.• The changes do not appear to be permanent
suggesting that the loss of cell lineage markers is insufficient for re-programming.
This may be advantageous for actually regenerative medicine for direct to neural conversion. Limiting the chance of transformation (cancer).
KDM5b has 2 roles during cell fate decisions
TCF3 p27Endo/Meso
KDM5b
Pro-Neural
??
Stem Cell factors
KDM5bKDM5b
Pro-proliferation
?
KDM5b mechanism• KDM5b regulates neural stem cells and potentially activity of
neurons through its target genes.• KDM5b is regulated through both its localization and the
components of its complex.• KDM5b is up-regulated in key populations after brain injury
Future Directions/Questions Is KDM5b localization and activity 2 separate signals? How do the components of the KDM5b complex effect post-
natal NSCs (i.e. differentiation, survival)? How does alteration of KDM5 activity (either positive or
negative) effect injury response of NSCs (in situ)
In vivo Model• KDM5b is an a pluripotency and a survival factor.• The choice appears to be based on upstream input, and
most likely cell type (i.e. stem cell vs. committed).• Taking advantage of this may allow for interrogation of
both long term injury and separately neural differentiation.
Small
peptide
inhibitors
1-7 days
RT-PCRChIP
Inject cells sub-dermal/IM or intra-cranial
Skin or other tissue from GFP+ mouse
Sol. factors
Small
peptide
inhibitors
Cells
Neural Blood and skin
AcknowledgementsLeanne Stalker Bijan DeySean Keating
Ramin ShiekhattarJoyce Papadimitriou-Taylor
Jonathan BramsonWilla LiaoAjapal Bhangu
Martin DoughtyMarc Meneghini
Ray TruantLise Munsie
Shawn LiWendy ZhuMarek GalkaHuadong Liu