Download DNA methylation

Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Dr. Péter Balogh and Dr. Péter Engelmann
Transdifferentiation and regenerative medicine –
Lecture 4
EPIGENETIC FACTORS
IN
TRANSDIFFERENTIATIO
N
TÁMOP-4.1.2-08/1/A-2009-0011
Epigenetics
• Epigenetics is a central molecular mechanism
in organism complexity
• Epigenetics studies the heritable changes in
genome function that occur without a change
in DNA sequence.
• Even if organisms have the same genotype,
depending on environmental changes, they can
have different phenotypes that are mediated
by epigenetics.
• 1942. C. Waddington
• Genetic code – epigenetic code / histone
code
• Main forms: DNA methylation, chromatin
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Chromatin remodelling and
histone modifications
Chromatin changes:
• Histons have altered structure or charge (“cis”)
• Histons have altered affinity to chromatin associated
proteins (“trans”)
Histon modifications: postranslational modifications of
histon globular domain
• Methylation mediated by protein arginine methyltransferase
(PRMT) and histone lysine methyltransferases (HKMT)
• Acetylation histone acetylases (HAT), and histone
deacetylases (HDAC)
• Ubiquitination
• Sumoylation
• Phosphorylation
• Citrullination
• ADP-ribosylation
Epigenetic gene regulation
of
stem cell genome
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DNA repair
DNA replication
DNA methylation
Chromatin remodelling
Non-coding RNAs
Histone modification
Chromatin package
Transcription regulatio
H2A
H3
H3
Methylation
Acetylation
H4
H4
H2B
H2B
Phosphorylation
Ubiquitylation
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DNA methylation
• Covalent modifications of cytosine nucleotides of CpG
dinucleotides
• Majority of methylated CpG dinucleotides are present
in heterochromatic region
• Promoter CpG islands
• DNA methylation is controlled by DNA
methyltransferases (Dnmts)
– Dnmt1: methylation of hemimethylated CpG sites
– Dnmt3a and Dnmt3b: de novo symmetric methylation of
DNA during embryonic development and
differentiation
• Function: methylation in mammals is required for gene
silencing
(NB. methylation of CpG islands on one X chromosome
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Detection procedures of DNA
methylation
• Affinity purification of methylated DNA
• Digestion with methylation-sensitive
restriction endonucleases
•
Bisulfite conversion
– Methylation specific PCR
– MethyLight (qPCR based approach)
– Microarray based approaches
– Bisulfite sequencing
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DNA methylation in stem
cells
Transcriptional activation
Passive demethylation
Transcriptional repression
Oct4
Sox2
Klf4
Transcriptional repression
DNMT1
Active demethylation
Transcriptional repression
Transcriptional activation
Klf4
Sox2 Oct4
Klf4
Sox2 Oct4
Demethylase
Symmetrically methylated DNAHemi methylated DNA
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DNA methylation profile of
ES cells
• Specific methylation profile can be compared
between stem cells and adults cells
• The pluripotency-associated genes Oct4 and
Nanog are largely unmethylated in ESCs and
induced pluripotent stem cells (iPSCs), and
methylated in differentiated cells
• In ES cells, high CpG promoters have low DNA
methylation levels, whereas low CpG
promoters have relatively high DNA
methylation levels
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Histone methylation
Histone methylation (mono-, di-, tri-) by
PRMT and HKMT enzymes can induce ON/OFF
signature for gene expression.
•
Methylation of lysine 4, 36, or 79 on
histone 3 (H3K4, H3K36, H3K79), lysine 20 of
H4 (H4K20), and lysine 5 of K2B (H2BK5)
accelerate gene transcription.
• Trimethylation of H3K9, H3K27, or H4K20
represents the inhibition of gene
expression.
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Histone methylation in stem
cells
DNA methylation data were examined in
conjunction with data on histone
modifications:
• H3K4me3 (which is generally considered to be
an activating mark) and H3K27me3 (a
repressive mark), it was found that genes
associated with H3K4me3 alone had the lowest
levels of promoter methylation (40%)
• 47% of the genes with the H3K4/H3K27
“bivalent” mark were methylated
• 70% of the genes with H3K27 alone were
methylated
• 87% of the genes carrying neither histone
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Histone acetylation
• Acetylation and deacetylation of lysine
residue in histone tails is mediated by
histone acetyl transferases (HATs) and
histone deacetylases (HDACs).
• 6 HAT complexes and 18 HDACs have been
identified in
mammals.
• Acetylation brings in a negative charge,
acting to neutralize the positive charge on
the histones and decreases the interaction
of the N termini of histones with the
negatively charged phosphate groups of DNA.
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Histone acetylation in stem
cells I
• Histone acetylation status of conserved
lysine residues in the amino- terminal of
histone H2A, H2B, H3, and H4 contributes to
transcriptional regulation.
• In general, an increase of histone
acetylation by HATs causes remodeling of
chromatin from a tightly to a loosely packed
configuration (euchromatin), which
subsequently leads to transcriptional
activation. Conversely, a decrease of
histone acetylation by HDACs results in a
condensed chromatin structure
(heterochromatin) and finally
transcriptional silencing
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Histone acetylation in stem
cells II
• Transcription factors Oct4 and Nanog
promoters are associated with activating
marks such as acetylation of H3 and H4 and
H3K4me3 in ES cells.
• Adult hippocampal-derived NSCs differentiate
predominantly into neurons, at the expense
of astrocytes and oligodendrocytes, when
treated by the antiepileptic and HDAC
inhibitor valproic acid (VPA) in vitro.
• VPA-mediated HDAC inhibition upregulates the
neuron-specific gene NeuroD, a neurogenic
basic helix-loop-helix TF, resulting in the
induction and suppression, respectively, of
neuronal and glial differentiation.
Ubiquitination and
sumoylation
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Ubiquitin :
• is a protein moiety that is covalently attached to proteins
via a series of enzymatic steps involving an ubiquitin
activating enzyme , a ubiquitin conjugating enzyme and
ubiquitin ligase .
•
Histone H2A was the first protein identified that served
as a substrate for ubiquitin.
•
Specific histon ubiquitination regulate histone
methylations.
SUMO (small ubiquitin-like modifier) proteins :
• covalently attaches to the residues of specific target
proteins and alters a number of various functions.
• Sumoylation counteracts ubiquitination and subsequent
proteosomal degradation via competition with the same
lysine residue of substrates.
•
Sumoylation regulatory mechanism is analogous to
Citrullination and
phosporylation
•
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Citrullination or deimination is a
posttranslational modification meaning the
change of arginine amino acid into
citrulline by peptidylarginine deiminases
(PADs).
• The conversion of arginine into citrulline
in histones can have important consequences
for the structure and function of proteins,
since arginine is positively charged at a
neutral pH, whereas citrulline is uncharged
which means protein folding changes.
• Histon phosphorylation occurs with H2A 139
serine residue in humans, which lead downstream protein target activations.
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Polycomb group factors
• Polycomb (PcG) proteins are transcriptional
regulators control the expression of many genes from
embryogenesis til adulthood.
• Polycomb group protein family can remodel chromatin
such that epigenetic silencing of genes take place.
• PcGs are evolutionarily conserved transcriptional
repressors, originally described as Hox gene
repressor.
• PcG proteins form three complexes: Polycomb
repressive complex 1 (PRC1), PRC2, and PhoRC.
• Two conserved repressor complexes (PRC1 and PRC2)
• PRC1 is composed by Cbx, Ring1, Phc, and
Bmi1/Mel18PRC2
• PRC2 is composed by EED, SUZ12, EZH2 factors
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Polycomb group proteins in
stem cells
• In ES cells pluripotency depends on the
activities of PcG and trithorax (TrxG)
complexes.
• PRC2 proteins as well as Ring1b or Ring1a
regulate the transcription of many genes in
ES cells.
• PRC2 catalyzes the di and trymethylation of
H3K27 motifs.
• PRC1 and PRC2 in ES cells targets promoters
of >2,000 genes, of which a large subset
overlaps with target genes of OCT4, NANOG,
and SOX2 transcription factors.
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Non-protein coding RNA:
Story I
mRNA: DNA -------- protein
tRNA, rRNA: structural, catalytic decoding
function
• 1998: Craig Mello, Andrew Fire: non-coding
RNA sequences, RNA interference in C.
elegans
• 2006: Nobel prize
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Non-protein coding RNA:
Story II
• The petal coloration of Petunia was supposed
to be changed by introducing the chalcon
sythase gene (coding a key enzyme for
coloration of petals) for overexpression.
• However it resulted a less colored or even
total
white
flowers
instead
of
darker
coloration. This supposed to be happened by
inhibition of the enzyme activity. Actually,
both
the
endogen
and
the
transgene
activitity was reduced in the white flowers.
This phenomenon was described as a corepression,
but
the
precise
molecular
mechanism remained unclear.
Non coding RNA, RNA
interference
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• siRNA(small interfering RNA): 21-22 nt long
dsRNA, gene silencing by siRNA, complementer
sequences, inhibitory proteins participate
in RNAi.
• miRNA (microRNA): 19-25 nt long, ssRNA
molecules, genomic, evolutionary conserved
• tncRNA (tiny non-coded RNA): newly
identified in C.elegans, 20-22 nt long
genomic RNA, function is unknown.
• smRNA (small modulatory RNA): described in
2004 from mice, neuron specific short dsRNA.
• PIWI associated small RNA (piRNA): 24-30 nt
long Drosophila, mammals, retrotransposons,
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miRNA role in stem cells I
• Dicer 1 and Dgcr8 enzymes are essential in
miRNA biogenesis, stem cells are defective
for these enzymes have pluripotency but
lower proliferation and differentiation
capacity.
• miRNAs encoded by Dlk1–Dio3 gene cluster are
also candidates for promoting reprogramming
because activation of imprinted Dlk1–Dio3
gene cluster is essential for generating
fully reprogrammed iPS cells, which are
functionally equivalent to ES cells.
• miRNAs belonging to the miR-290 and miR- 302
clusters are expressed in both human and
mouse ES cells.
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miRNA and stem cell
differentiation
ES/iPS Cells
Somatic Cells
miR-470 miR-134
inhibitor inhibitor
ESCC
miRNAs
Myc induced
miR-92b miRNAs
miR-296
ESCC
miR-520
miR-470
Myc induced
cluster miR-200
miR-145 inhibitor inhibitor miRNAs
miRNAs
miR-141
miR-296
+
miR-520
miR-429
p53
+
?
cluster
+
miR-17-92 cluste
miR-134
+
miR-92b
miR-145
p21 +
?
?
?
+
?
?
?
+
Self-renewal
and
pluripotency
Reprogramming by Sox2, Oct4, Klf4, and c-Myc or Sox2, Oct4, Nanog, and Lin28
let-7
?
+
miR-125 inhibitor
let-7 inhibitor
miR-125
miR-24-1
miR-23b
?
miR-21
Inhibitors of miR-24-1, miR-23b, miR-21
+
TGF-beta signalling
Active Dlk1-Dio3 gene cluster
+
HDAC inhibitor
CpG
Inactive Dlk1-Dio3 gene
?
miRNAs coded by Dlk1-Dio3 gene cluster
Oct4
+
?
Dnmt knock-down/Dnmt inhibitors
Dnmt 3a and 3b miR-29b
mCpG
Oct4
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miRNA role in stem cells II
• miR-124a is expressed predominantly in neural
tissues and has been shown to participate in the
in vitro differentiation of NSCs into neurons by
mediating degradation of non-neuronal gene
transcripts.
• Both miR-124 and miR-9 promotes neuronal
differentiation, while their down regulation has
the opposite effect.
• miR-294 exhibited the greatest effect on
reprogramming and increased efficiency of iPS
cell generation from 0.01–0.05% to 0.4–0.7%.
Additionally, miR-294 increased the kinetics of
Sox2, Oct4, and Klf4 mediated reprogramming.
• Inhibiting the activity of both miR-125 and
let-7 miRNAs may result in additional beneficial
Cross-talk between genetic
and epigenetic regulators
in ESCs I
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• Transcription factors and epigenetic factors
interact on multiple dimensions.
• Key pluripotency factors such as Oct4 and
Nanog interact physically with epigenetic
regulators to maintain ESCs in an
undifferentiated state.
• Oct4, Sox2 and Nanog co-occupy many of the
PRC2 target genes, which are enriched for
developmental/differentiation genes.
• Through protein interactions, transcription
factors such as Oct4 may directly recruit
and provide target specificity for
repressive PcG proteins such as Ring1b.
Cross-talk between genetic
and epigenetic regulators
in ESCs II
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• Transcription factors and epigenetic factors
interact to modulate the ESC regulatory
network.
• Several ESC-specific epigenetic factors are
regulated by the core ESC transcription
factors.
• Oct4 activates the expression of histone
demethylases, Jmjd1a and Jmjd2c, which in
turn modify H3K9 methylation and regulate
the expression of Tcl1 and Nanog.
Therapeutical
considerations
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• Epigenetic changes are reversible upon
administration of Dnmt inhibitors and
histone deacetylase inhibitors (HDACi)
• Dnmt inhibitors such as azacitidine (5-azacytidine), decitabine (5-aza-2`deoxycytidine) has been approved by FDA in
cancer treatment.
• HDACi such as hydralazine and magnesium
valproate are capable to sensitize tumor
cells to chemotherapy in patients with solid
tumors.
• How about cancer stem cells??
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Summary
• Life of an individual is not only defined by
her/his genome, but also by the numerous
epigenomes, with different epigenomes being
generated through development.
•
Epigenomes react to environmental influence
including maternal care, diet, exposure to
toxins and xenobiotics, and epigenetic
responses to environmental stimuli may have
long-term consequences, even affecting
future generations.
•
At various level epigenetics affects the
stem cell niche, and they build up multiple
levels of regulatory circuits in progenitor