KDM5 epigenetic modifiers as a focus for drug discovery

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