Epigenetic factors in transdifferentiation

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

2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. PéterBalogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 4 Epigeneticfactorsintransdifferentiation

3. Epigenetics • Epigeneticsis a central molecular mechanism in organism complexity • Epigeneticsstudiesthe heritable changes in genome functionthat occur without a change inDNA sequence. • Even if organisms have the same genotype, dependingonenvironmental changes, they canhave different phenotypes that are mediated by epigenetics. • 1942. C. Waddington • Geneticcode – epigeneticcode / histonecode • Main forms: DNA methylation, chromatinremodelling, histonemodifications and non-coding RNA sequences.

4. Chromatinremodelling and histonemodifications • Chromatinchanges: • Histonshave altered structureorcharge (“cis”) • Histonshave altered affinitytochromatinassociatedproteins (“trans”) • Histonmodifications: postranslationalmodifications of histonglobulardomain • Methylationmediatedby protein argininemethyltransferase (PRMT) and histonelysinemethyltransferases (HKMT) • Acetylationhistoneacetylases (HAT), and histonedeacetylases (HDAC) • Ubiquitination • Sumoylation • Phosphorylation • Citrullination • ADP-ribosylation

5. Epigenetic gene regulation ofstem cell genome DNA repair DNA replication DNA methylation Chromatin remodelling Non-coding RNAs Histone modification H2A Methylation H2B H3 H4 Acetylation Chromatin package Transcription regulation H2B Phosphorylation H3 H4 Ubiquitylation

6. DNA methylation • Covalentmodifications of cytosinenucleotidesofCpGdinucleotides • Majority of methylatedCpGdinucleotidesarepresentinheterochromaticregion • PromoterCpGislands • DNA methylation is controlledbyDNA methyltransferases (Dnmts) • Dnmt1: methylation of hemimethylatedCpGsites • Dnmt3a and Dnmt3b: de novosymmetricmethylation of DNA duringembryonicdevelopment and differentiation • Function: methylationinmammals is requiredforgenesilencing • (NB. methylation of CpGislandsonone X chromosomeresults X- chromosomeinactivation – genomicimprinting).

7. 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

8. DNA methylation in stem cells Transcriptional activation Passive demethylation Transcriptional repression Sox2 Oct4 Klf4 Transcriptional repression DNMT1 Active demethylation Transcriptional repression Transcriptional activation Klf4 Klf4 Oct4 Oct4 Sox2 Sox2 Demethylase Symmetrically methylated DNA Hemi methylated DNA

9. DNA methylationprofile of ES cells • Specificmethylationprofilecan be comparedbetweenstemcells and adultscells • 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, highCpG promoters have low DNA methylation levels, whereaslow CpG promoters have relatively high DNA methylationlevels

10. Histonemethylation • Histonemethylation(mono-, di-, tri-) by PRMT and HKMT enzymescaninduce ON/OFF signatureforgeneexpression. • Methylation of lysine 4, 36, or 79 on histone3 (H3K4, H3K36, H3K79), lysine 20 of H4 (H4K20), and lysine 5 of K2B (H2BK5)accelerategenetranscription. • Trimethylation of H3K9, H3K27, or H4K20 representstheinhibition of geneexpression.

11. Histonemethylationinstemcells • 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 mark were methylated

12. Histoneacetylation • Acetylation and deacetylation of lysine residue in histone tails is mediated by histone acetyl transferases (HATs) and histonedeacetylases (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.

13. HistoneacetylationinstemcellsI • Histoneacetylation status of conservedlysineresiduesintheamino-terminalofhistone H2A, H2B, H3, and H4 contributestotranscriptionalregulation. • In general, an increase of histoneacetylation by HATs causes remodeling of chromatinfrom a tightly to a loosely packed configuration(euchromatin), which subsequently leads totranscriptionalactivation. Conversely, a decrease of histone acetylation by HDACs results in a condensed chromatinstructure (heterochromatin) and finally transcriptionalsilencing

14. HistoneacetylationinstemcellsII • Transcriptionfactors Oct4 and Nanog promoters are associated with activating markssuch asacetylation of H3 and H4 and H3K4me3in ES cells. • Adult hippocampal-derived NSCs differentiate predominantlyinto neurons, at the expense of astrocytesandoligodendrocytes, when treated by the antiepileptic and HDAC inhibitor valproicacid (VPA) in vitro. • VPA-mediated HDAC inhibitionupregulates the neuron-specific gene NeuroD, a neurogenicbasic helix-loop-helix TF, resulting in theinduction and suppression, respectively, of neuronal and glialdifferentiation.

15. Ubiquitination and sumoylation • Ubiquitin: • is a protein moiety that is covalently attached toproteins via a series of enzymatic steps involving an ubiquitin activatingenzyme , a ubiquitinconjugatingenzyme and ubiquitinligase . • Histone H2A was the first protein identifiedthat served as a substrate for ubiquitin. • Specifichiston ubiquitinationregulatehistonemethylations. • 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 ofconserved enzymes.

16. Citrullination and phosporylation • Citrullinationordeimination is a posttranslationalmodificationmeaningthechange of arginineaminoacidintocitrullinebypeptidylargininedeiminases (PADs). • The conversion of arginine into citrullineinhistones can have important consequences for the structure and function of proteins, since arginine is positively charged at a neutral pH, whereas citrulline is unchargedwhichmeans protein foldingchanges. • Histonphosphorylationoccurswith H2A 139 serine residueinhumans, which lead down-stream protein targetactivations.

17. Polycombgroupfactors • Polycomb (PcG) proteinsaretranscriptional regulators controltheexpression of manygenesfromembryogenesistiladulthood. • Polycombgroup protein familycanremodelchromatinsuchthatepigeneticsilencing of genestakeplace. • PcGsareevolutionarilyconservedtranscriptionalrepressors, originallydescribedasHoxgenerepressor. • PcG proteins form three complexes: Polycomb repressive complex 1 (PRC1), PRC2, and PhoRC. • Twoconservedrepressorcomplexes (PRC1 and PRC2) • PRC1 is composedbyCbx, Ring1, Phc, and Bmi1/Mel18PRC2 • PRC2 is composedby EED, SUZ12, EZH2 factors

18. Polycombgroupproteinsinstemcells • In ES cells pluripotency dependsontheactivities of PcG and trithorax (TrxG) complexes. • PRC2 proteins as well as Ring1b or Ring1a regulate the transcriptionof many genes in ES cells. • PRC2 catalyzesthe di and trymethylation of H3K27 motifs. • PRC1 and PRC2 in ES cells targets promoters of >2,000 genes, ofwhich a large subset overlaps with target genes of OCT4, NANOG, and SOX2 transcriptionfactors.

19. Non-protein coding RNA: Story I • mRNA: DNA -------- protein • tRNA, rRNA: structural, catalyticdecodingfunction • 1998: Craig Mello, Andrew Fire: non-coding RNA sequences, RNAinterferenceinC. elegans • 2006: Nobel prize

20. Non-protein coding RNA: Story II • The petalcoloration of Petuniawassupposedto be changedbyintroducingthechalconsythasegene (coding a keyenzymeforcoloration of petals) foroverexpression. • Howeveritresulted a less coloredoreventotalwhiteflowersinstead of darkercoloration. Thissupposedto be happenedbyinhibition of theenzymeactivity.Actually, boththeendogen and thetransgeneactivititywasreducedinthewhiteflowers. Thisphenomenonwasdescribedas aco-repression, buttheprecisemolecularmechanismremainedunclear.

21. Non coding RNA, RNAinterference • siRNA(smallinterfering RNA):21-22 ntlongdsRNA, genesilencingbysiRNA, complementersequences, inhibitoryproteinsparticipateinRNAi. • miRNA (microRNA): 19-25 ntlong, ssRNAmolecules, genomic, evolutionaryconserved • tncRNA (tinynon-coded RNA):newlyidentifiedinC.elegans, 20-22 ntlonggenomic RNA, function is unknown. • smRNA(smallmodulatory RNA): describedin 2004 frommice, neuron specificshortdsRNA. • PIWI associatedsmall RNA (piRNA): 24-30 ntlong Drosophila, mammals, retrotransposons, against mobile geneticelements.

22. miRNAroleinstemcellsI • Dicer 1 and Dgcr8 enzymesareessentialinmiRNAbiogenesis, stemcellsaredefectivefortheseenzymeshavepluripotencybutlowerproliferation and differentiationcapacity. • 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.

23. miRNA and stemcelldifferentiation ES/iPSCells SomaticCells miR-470 inhibitor miR-134 inhibitor Mycinduced miRNAs ESCC miRNAs miR-92b miR-296 inhibitor ESCC miRNAs miR-520 cluster miR-470 Mycinduced miRNAs miR-200 miR-141 miR-429 miR-17-92 cluster miR-145 inhibitor miR-296 + miR-520 cluster p53 + ? + miR-134 + miR-92b + miR-145 p21 ? ? ? + ? ? ? + Self-renewaland pluripotency Reprogrammingby Sox2, Oct4, Klf4, and c-Mycor 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-betasignalling Active Dlk1-Dio3 genecluster Inactive Dlk1-Dio3 genecluster + ? HDAC inhibitor miRNAscodedby Dlk1-Dio3 genecluster Oct4 Oct4 CpG mCpG ? + Dnmtknock-down/Dnmtinhibitors Dnmt 3a and 3b miR-29b

24. miRNAroleinstemcellsII • miR-124a is expressed predominantly in neural tissuesand has been shown to participate in the in vitrodifferentiation of NSCs into neurons by mediating degradation of non-neuronalgenetranscripts. • Both miR-124 and miR-9 promotesneuronaldifferentiation, while their downregulation has the oppositeeffect. • miR-294 exhibited thegreatest effect on reprogramming and increased efficiency of iPS cellgeneration from 0.01–0.05% to 0.4–0.7%. Additionally, miR-294increased the kinetics of Sox2, Oct4, and Klf4 mediated reprogramming. • Inhibiting the activity of both miR-125 andlet-7 miRNAs may result in additional beneficial effects duringreprogramming, due to robust activation of Lin28 expression.

25. Cross-talk between genetic and epigenetic regulators in ESCsI • 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.

26. Cross-talk between genetic and epigenetic regulators in ESCsII • 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 histonedemethylases, Jmjd1a and Jmjd2c, which in turn modify H3K9 methylation and regulate the expression of Tcl1 and Nanog.

27. Therapeuticalconsiderations • Epigeneticchangesarereversibleuponadministration of Dnmtinhibitorsand histonedeacetylaseinhibitors(HDACi) • Dnmtinhibitorssuchasazacitidine (5-aza-cytidine), decitabine (5-aza-2`-deoxycytidine) has beenapprovedby FDA incancertreatment. • HDACisuchashydralazine and magnesiumvalproatearecapabletosensitize tumor cellstochemotherapyinpatientswithsolidtumors. • Howaboutcancerstemcells??

28. Summary • Life of an individual is not only defined by her/his genome,but also by the numerous epigenomes, with differentepigenomes being generated through development. • Epigenomes react to environmental influence includingmaternal care, diet, exposure to toxins and xenobiotics, andepigenetic responses toenvironmental stimuli may havelong-term consequences, even affecting future generations. • Atvariouslevelepigeneticsaffectsthestemcellniche, and theybuildupmultiplelevels of regulatorycircuitsinprogenitorcells.