Epigenetic Control

of Skin Regeneration

and Aging

or

How Chromatin Regulators

Orchestrate Skin Functions

Prof. Vladimir Botchkarev, MD, PhD

Centre for Skin Sciences, University of Bradford, UK

Department of Dermatology, Boston University, USA

Skin development:

What control the establishment

of lineage-specific differentiation programs?

Duijf & van Bokhoven, Drug Discovery Today, 2005

Signaling pathways (Wnt, Bmp, Hh, Notch, Fgf, NF-kB, etc.)

Transcription factors (p63, Mitf, Atoh1, etc.)

Epigenetic regulators (Dnmts, Hdacs, ATP-dependent

and higher-order chromatin remodelers, etc.)

Molecular control of cell differentiation:

Integration of several levels of regulation

Epigenetic is the field of biology studying phenotypic

inheritance in cells and organisms that does not result

from the changes in the nucleotide sequence of DNA

What is Epigenetics?

Conrad H. Waddington

(1905-1975)

The definition of 'epigenetics' was introduced by British scientist Conrad H. Waddington in 1942.

Conrad Waddington described epigenetics as “…the interactions of genes with their

environment that bring the phenotype into

being”.

Epigenetic mechanisms control

variability of skin phenotypes

and distinct responses to environment

Epigenetic mechanisms

regulate stability and flexibility of the genome

Felsenfeld and Groudine Science, 2003

Chromatin is DNA-protein complex

organized into several levels

Levels of epigenetic regulation

in the interphase nucleus

DNA methylation and hydroxymethylation

Post-translational histone modifications

Non-coding RNA-dependent mechanisms

ATP-dependent chromatin remodeling

Higher-order chromatin remodeling and

nuclear compartmentalization of active and repressed

genes and chromatin domains

DNA methylation and hydroxymethylation

are powerful mechanisms regulating gene expression

DNMTs

Gene Silencing

Methylated DNA binds

Methyl-DNA binding proteins

that recruit repressive complexes

to the methylated DNA

5-hydroxymethyl-

cytosine

TETs

OH

Gene

Activation

…

Basal epidermal keratinocytes

Keratins 5/14, Desmosomal proteins, E-cadherin

Cell proliferation

Immediate suprabasal epidermal keratinocytes

Onset of terminal differentiation, Keratins 1/10

Terminally differentiated keratinocytes

Involucrin, Loricrin, Filaggrin, Cornifin,

Transglutaminases

Molecular signature of epidermal layers:

Structural proteins

DNA methylation promotes cell proliferation

and prevents premature activation of

the terminal differentiation genes in the epidermis

Khavari’s lab (Stanford):

Inactivation of DNMT1 results in epidermal thinning,

premature onset of expression of terminal differentiation genes,

as well as in decrease of cell proliferation associated

with increased expression of cell cycle inhibitors

p15/INK4B and p16/INK4A

Sen et al. Nature 2010

Chuong’s lab (USC):

Ablation of Dnmt1 leads to decrease of cell proliferation

in the hair follicles and progressive hair loss

Li et al. J Invest Dermatol 2012

Levels of epigenetic regulation

in the interphase nucleus

DNA methylation and hydroxymethylation

Post-translational histone modifications

Non-coding RNAs

ATP-dependent chromatin remodeling

Higher-order chromatin remodeling and

nuclear compartmentalization of active and repressed

genes and chromatin domains

Post-translational covalent modifications

of histone proteins are crucial regulators

of gene activation and silencing

Polycomb-repressive complex

is a key regulator of H3K27 methylation status

Eed

Ezh2

Rbbp4

PRC2

H3K27me3

PHC1 Cbx4

Ring1

Bmi1

PRC1

Ubi

me

me

H2AK119ub1

PRC1

Ring1, Rnf2

Cbx2, Cbx4, Cbx6, Cbx7, Cbx8

Bmi1, Pcgf1, Pcgf2, Pcgf6

Phc1, Phc2, Phc3

PRC2 Ezh2, Ezh1

Suz12 Eed

Rbbp4, Rbbp7

Phf1, Mtf2, Phf19, Jarid2

Gene OFF

Polycomb-dependent H3K27 methylation

prevents premature activation of terminal differentiation

program in the basal epidermal layer

Fuchs’s lab (Rockefeller):

Ablation of Ezh2 methytransferase results in

premature onset in expression

of terminal differentiation-associated genes

Ezhkova et al. Cell 2009

Ezhkova’s lab (Mount Sinai):

Polycomb complex restricts differentiation of epidermal

progenitor cells by repressing the transcription factor Sox2,

which, in turn, promotes differentiation of the progenitors

towards Merkel cell lineage

Bardot et al. EMBO J 2013

Cbx4 is expressed in both basal and suprabasal

epidermal cells in developing and postnatal skin

Andrei Mardaryev Research Investment Lecturer,

Centre for Skin Sciences,

University of Bradford

Cbx4 KO mice show premature keratinocyte

differentiation during epidermal development

De-repression of neuronal genes

in the epidermis of Cbx4 KO mice

Fo

ld c

han

ge

no

rmalised

to

Gap

dh

0

2

4

6

8

10

Neurog3 En2 Nefl Mobp Olig2 Lhx4

WT

Cbx4 KO

*

*

WT Cbx4KO

Nefl Krt14

*

E16.5

Cbx4 is a direct target for p63 transcription factor

in epidermal keratinocytes

Cbx4 is a part of p63-regulated programme

that maintains epithelial lineage identity

and cell proliferation in the developing epidermis

Mardaryev et al. J Cell Biol, 2016

Levels of epigenetic regulation

in the interphase nucleus

DNA methylation and hydroxymethylation

Post-translational histone modifications

Non-coding RNAs

ATP-dependent chromatin remodeling

Higher-order chromatin remodeling and

nuclear compartmentalization of active and repressed

genes and chromatin domains

Cell Nucleus is a Site of Storage and Replication

of Genetic Material

Botchkarev et al, J Invest Dermatol, 2012

Mike Fessing Lecturer,

CSS, University of Bradford

Andrei Mardaryev Research Investment Lecturer,

CSS, University of Bradford

Andrey Sharov Assistant Professor

Boston University

Nuclear architecture is markedly re-organized

during epidermal differentiation

Epidermal differentiation:

1) Decrease of nuclear volume

2) Spreading of heterochromatin

Gdula et al, J Invest Dermatol, 2012

3) Fusion and decrease in number

of nucleoli

Chr 3 Chr 11 Chr 15

Epidermal

Differentiation

Complex

Keratin

Cluster 1

(keratins 1, 10, 14, 15,

16, 17, 19)

Keratin

Cluster 2

(keratins 5, 6, 8, 18)

Majority of the genes encoding

keratinocyte-specific structural proteins

are localized on chromosomes 3, 11, and 15

Epidermal differentiation complex,

mouse chromosome 3

93.2-96.4 Mb

Rps27

90

Gene density on chromosome 3

Martin, Patel & Segre Genome Res 2004

Increase in transcription activity within EDC

is accompanied by the developmentally-regulated

relocation from the peripheral to internal part

of chromosomal territory 3

Mike Fessing Lecturer,

CSS, University of Bradford

Andrei Mardaryev Research Investment Lecturer,

CSS, University of Bradford

Fessing/Mardaryev et al. J Cell Biol 2011

Chr 3

EDC

EDC and Lor relocation to nuclear interior

is a lineage- and differentiation stage-specific process

Dermal fibroblasts show peripheral positioning

of the EDC and Lor gene Sox9+ bulge stem cells

show peripheral positioning

of the Lor gene

p63 is essential for

early epidermal development

p63 is a homolog of p53 transcription factor

p63 knockout mice lack stratified epidermis

and show single-layered K5/14-negative epithelium (Mills et al. Nature, 1999; Yang et al. Nature, 1999)

p63 -/-

p63 knockout mice also show hypoplasia of thymus

and alterations in T-lymphocyte development (Candy, PNAS, 2007)

p63 -/- skin epithelium shows alterations in

expression of genes involved in chromatin

organization and remodeling

Mills et al. Nature 1999

Fessing/Mardaryev et al. J Cell Biol 2011

p63 directly regulates Brg1 and Satb1

in primary keratinocytes (ChIP assay)

Fessing/Mardaryev et al. J Cell Biol 2011

Mardaryev et al. Development 2014

p63 and Brg1 regulate EDC relocation

towards nuclear interior

Chr 3

EDC

p63 KO, Brg1 KO

Brg1 is ATP-dependent chromatin remodeler

required for gene activation

during epidermal barrier formation

Brg1 is a part of molecular circuit that integrates extracellular signaling

(Shh), transcriptional regulation (NF-kB, Gli) and chromatin remodeling in

hair follicle stem cells

Xiong et al. Dev Cell, 2013

Brg1 is required for late steps

of terminal keratinocyte

differentiation and epidermal

barrier formation

Indra et al. Development, 2005

Arup Indra (Oregon State):

AT-rich binding protein Satb1

controls establishing specific conformations

in a number of tissue-specific gene loci

TH-2

cytokine

locus

Cai et al. Nature Genet 2006

Terumi Kohwi-

Shigematsu

Berkeley

DNA

Fessing/Mardaryev et al. J Cell Biol 2011

Genetic Satb1 ablation: Alterations in the EDC conformation and gene expression

p63 and epigenetic machinery

operate in concert

in regulating keratinocyte differentiation

How the Epigenetic Machinery

Contributes to Skin Aging?

p63 deficiency results in shortened

life-span and accelerated aging

associated with enhanced expression

of the senescence markers

Keyes et al. Genes&Dev, 2005

p63-regulated gene expression programs

are altered during aging

Alea Mills Cold Spring Harbor

Laboratory

TAp63 induces senescence

and suppresses tumorigenesis in vivo

Guo et al. Nature Cell Biol, 2011

Future directions of epigenetic research:

How to re-activate “good genes”

and repress “bad genes” in skin cells ?

DNMT inhibitors:

Azacytidine, Decitabine

Myelodisplastic Syndrome

HDAC inhibitors:

Vorinostat, Romodepsin

Cutaneous T-cell Lymphoma

EZH2 Inhibitors:

EPZ7438

Non-Hodgkin Lymphoma

International Symposium“Epigenetic Control of Skin Development

and Regeneration”April 2nd – 3rd, 2012

Centre for Skin Sciences, University of Bradford, UK

Organizers: Vladimir Botchkarev

Mike FessingNatalia Botchkareva

Desmond Tobin

Speakers:

Wendy BickmoreChen-Ming ChuongShaun CowleyElena EzhkovaMichaela FryeDenis HeadonTerumi Kohwi-ShigematsuSarah MillarAndrei PanteleyevAndrey SharovKevin WangRui Yi

Scientific Sessions:Biology of the Keratinocyte Nucleus

DNA & Histone ModificationsHigh-Order Chromatin Remodelling

Non-Coding and micro-RNAsBioinformatic Analyses of Epigenome

Epigenetics Drugs and Skin Diseases

Abstract Submission Deadline: December 15th, 2011

Selected abstracts will be invited for oral presentations

Registration Fees: Academic researchers – 150 Euro Industrial researchers – 300 Euro

PhD students – waived

Contact information: www.skin.brad.ac.uk

2nd International Symposium

“Epigenetic Regulation of Skin Regeneration,

Ageing and Disease”

Bradford

March 17th-19th, 2016

Terumi Kohwi-Shigematsu

(Berkeley)

Guoliang Xu

(Shanghai)

Salvador Aznar-Benitah

(Barcelona)

University of Bradford

Andrei Mardaryev

Mike Fessing

Krzysztof Poterlowisz

Jana Rudolf

Igor Malashchuk

Boston University

Andrey Sharov

Vladimir Emelianov

Tanya Sharova