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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
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
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 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
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 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