Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

EPIGENETIC FACTORS IN TRANSDIFFERENTIATION

Dr. Péter Balogh and Dr. Péter EngelmannTransdifferentiation and regenerative medicine – Lecture 4

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

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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

remodelling, histone modifications and non-coding RNA sequences.

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Chromatin remodelling and histone modificationsChromatin 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

TÁMOP-4.1.2-08/1/A-2009-0011Epigenetic gene regulation ofstem cell genomeDNA replication DNA repair

Chromatin package Transcription regulation

Histone modification

Chromatin remodelling

DNA methylation

Non-coding RNAs

Methylation Acetylation UbiquitylationPhosphorylation

H3

H4

H2B

H2AH3

H4

H2B

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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 results X- chromosome inactivation – genomic imprinting).

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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 cellsPassive demethylation

Active demethylation

Transcriptional repression

Transcriptional repression

DNMT1

Klf4Oct4

Sox2

Transcriptional activationTranscriptional repression

Klf4 Oct4Sox2

Demethylase

Klf4 Oct4Sox2

Symmetrically methylated DNA Hemi methylated DNA

Transcriptional activation

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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 cellsDNA 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 mark were

methylated

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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.

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Ubiquitination and sumoylationUbiquitin :• 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 ubiquitin-conjugation system, but sumoylation is regulated by a different set of conserved enzymes.

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Citrullination and phosporylation • 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 down-stream 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 instem 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 ImRNA: DNA -------- proteintRNA, 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 co-repression, but the precise molecular mechanism remained unclear.

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Non coding RNA, RNA interference• 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, against mobile genetic elements.

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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

+

+

miR-520cluster

miR-92b

Myc inducedmiRNAs

ESCCmiRNAs

Self-renewaland

pluripotency

miR-296

miR-470

miR-134

HDAC inhibitor miRNAs coded by Dlk1-Dio3 gene cluster

Dnmt knock-down/Dnmt inhibitors Dnmt 3a and 3b miR-29b

TGF-beta signalling Inhibitors of miR-24-1, miR-23b, miR-21

Reprogramming by Sox2, Oct4, Klf4, and c-Myc or Sox2, Oct4, Nanog, and Lin28

let-7 inhibitormiR-125 inhibitor

miR-125

miR-145 inhibitor

miR-145

p53

p21

miR-200miR-141miR-429miR-17-92 cluster

ES/iPS Cells Somatic Cells

Active Dlk1-Dio3 gene cluster Inactive Dlk1-Dio3 gene cluster

miR-24-1miR-23bmiR-21

let-7

ESCCmiRNAs

+

miR-470inhibitor

?

miR-296inhibitor

?

miR-134inhibitor

?

miR-92b

?

miR-520cluster

?

?

?

+ ?

?

?

Myc inducedmiRNAs

?

+

mCpG Oct4CpG 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 effects during reprogramming, due to robust activation of Lin28 expression.

TÁMOP-4.1.2-08/1/A-2009-0011Cross-talk between genetic and epigenetic regulators in ESCs I• 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.

TÁMOP-4.1.2-08/1/A-2009-0011Cross-talk between genetic and epigenetic regulators in ESCs II• 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.

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Therapeutical considerations

• Epigenetic changes are reversible upon administration of Dnmt inhibitors and histone deacetylase inhibitors (HDACi)

• Dnmt inhibitors such as azacitidine (5-aza-cytidine), 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 cells.