Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming

Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies

p68/DdX5 supports β-Catenin & RNAP II during androgen receptor mediated transcription in prostate cancer

The DEAD box RNA helicase p68 (Ddx5) is an important androgen receptor (AR) transcriptional co-activator in prostate cancer (PCa) and is over-expressed in late stage disease. β-Catenin is a multifunctional protein with important structural and signalling functions which is up-regulated in PCa and similar to p68, interacts with the AR to co-activate expression of AR target genes. Importantly, p68 forms complexes with nuclear β-Catenin and promotes gene transcription in colon cancer indicating a functional interplay between these two proteins in cancer progression. In this study, we explore the relationship of p68 and β-Catenin in PCa to assess their potential co-operation in AR-dependent gene expression, which may be of importance in the development of castrate resistant prostate cancer (CRPCa). We use immunoprecipitation to demonstrate a novel interaction between p68 and β-Catenin in the nucleus of PCa cells, which is androgen dependent in LNCaP cells but androgen independent in a hormone refractory derivative of the same cell line (representative of the CRPCa disease type). Enhanced AR activity is seen in androgen-dependent luciferase reporter assays upon transient co-transfection of p68 and β-Catenin as an additive effect, and p68-depleted Chromatin-Immunoprecipitation (ChIP) showed a decrease in the recruitment of the AR and β-Catenin to androgen responsive promoter regions. In addition, we found p68 immunoprecipitated with the processive and non-processive form of RNA polymerase II (RNAP II) and show p68 recruited to elongating regions of the AR mediated PSA gene, suggesting a role for p68 in facilitating RNAP II transcription of AR mediated genes. These results suggest p68 is important in facilitating β-Catenin and AR transcriptional activity in PCa cells

Γενετικά και επιγενετικά ρυθμιστικά δίκτυα στα εμβρυϊκά βλαστικά κύτταρα του ποντικού

In the past decade, numerous studies demonstrated that both microRNAs (miRNAs) and Promyelocytic Leukemia Protein (PML) were central regulators of different biological processes including cell proliferation, apoptosis and tumorigenesis. Emerging research links miRNAs as well as PML with the regulation of stem cells maintenance. Several miRNAs were reported to sustain embryonic stem cells (ESCs) identity (miR-290-295), while others to induce ESC differentiation (miR-34a, miR-145). At the same time, recent reports using p19 embryonal carcinoma cells and ESCs showed that Nanog and Oct4 – the core pluripotent regulators - associate with PML protein. Moreover, PML was described as a regulator of metabolic pathways in stem cell compartments (hematopoietic stem cells-HSCs) and as an essential component in leukemia-initiating cells maintenance. In an effort to elucidate the molecular mechanism underlying the contribution of PML and miRNAs in stemness phenotype, we examined their role in mESCs.In the first part of this work, we present four new miRNAs (miR-16-1, miR-191, miR-23a, miR-421), which are differentially expressed upon differentiation. miR-191 and miR-16-1 are highly expressed in ESCs and inhibits Activin-Nodal signaling, leading to the suppression of mesoderm and endoderm differentiation. miR-23a, which is also down-regulated in the differentiated state, represses differentiation towards the endoderm and ectoderm lineages. In contrast, miR-421 was characterized as a differentiation-associated regulator that targets the core pluripotency transcription factor Oct4 and the BMP-signaling pathway to promote endoderm and ectoderm differentiation. Our results uncover a regulatory network between the studied miRNAs and both branches of TGF-β/BMP signaling pathways unveiling their importance for ESC lineage decisions. In the second part of this thesis, we show that PML positively correlates with the undifferentiated state and is a vital regulator of ESC pluripotency. PML knockdown induces up-regulation of multi-lineage differentiation markers and produces enhanced differentiation towards mesodermal lineage, while ectopic expression of PML prevents ESC differentiation in vitro and maintains stem cell phenotypes. Through transcriptome analysis of ESCs WT and ESC PML KD, we identified a large number of differentially expressed genes. Specifically, a group of deregulated genes is involved to the transition from naïve to primed pluripotent state, while a great number of genes associate with both signaling pathways essential for ESC identity as well as cell cycle. Additionally, reprogramming of Pml-/-mouse embryonic fibroblasts (MEFs) showed significantly lower efficiency compared to MEFs WT, highlighting PML as a pivotal mediator of somatic cell reprogramming. We speculate that the potential mechanism is based on regulation of EMT through TGF-β signaling. Our findings reveal that PML acts as an essential regulator in naïve and primed pluripotency as well as somatic cell reprogramming.Τα τελευταία χρόνια, πληθώρα ερευνητικών εργασιών κατέδειξε ότι τόσο τα μικρά-RNAs όσο και η πρωτεΐνη προμυελοκυταρικής λευχαιμίας (ΠΠΛ) διαδραματίζουν σημαντικό ρόλο σε πολλές κυτταρικές διαδικασίες, συμπεριλαμβανομένου του πολλαπλασιασμού, της απόπτωσης και της ογκογένεσης. Παράλληλα, ο ρόλος τους έχει συσχετιστεί με τη ρύθμιση των βλαστικών κυττάρων. Συγκεκριμένα, έχει δειχθεί ότι πολλά μικρά-RNAs συμβάλουν στη διατήρηση των εμβρυικών βλαστικών κυττάρων (ΕΒΚ) (miR-290-295), ενώ άλλα προάγουν τη διαφοροποίηση αυτών (miR-34a, miR-145). Επιπρόσθετα, έρευνες με τη χρήση κυττάρων εμβρυικού καρκινώματος και ΕΒΚ ανέδειξαν την αλληλεπίδραση δύο βασικών ρυθμιστών της πλειοδυναμίας -Oct4 και Nanog- με τη ΠΠΛ, η οποία με τη σειρά της έχει χαρακτηρισθεί ως ρυθμιστής των μονοπατιών μεταβολισμού σε βλαστικά συστήματα (αιμοποιητικά βλαστικά κύτταρα) και ως βασικό στοιχείο της διατήρησης των λευχαιμικών αρχέγονων κυττάρων. Η παρούσα διδακτορική διατριβή είχε ως στόχο τη κατανόηση του μοριακού μηχανισμού ρύθμισης των ΕΒΚ τόσο από τα μικρά-RNAs όσο και από τη ΠΠΛ.Στο πρώτο μέρος της διατριβής, παρουσιάζονται 4 νέα μικρά-RNAs (miR-16-1, miR-191, miR-23a, miR-421), τα οποία εκφράζονται ποικιλοτρόπως κατά τη διαφοροποίηση. Συγκεκριμένα, τα miR-16-1 και miR-191 εκφράζονται σε υψηλά επίπεδα στα ΕΒΚ και καταστέλλουν το σηματοδοτικό μονοπάτι Activin-Nodal, παρεμποδίζοντας τη μεσοδερμική και ενδοδερμική διαφοροποίηση. Το miR-23a, του οποίου η έκφραση μειώνεται σημαντικά κατά τη διαφοροποίηση, μετριάζει τη κατεύθυνση προς την ενδοδερμική και εκτοδερμική στιβάδα. Αντίθετα, το miR-421 στοχεύει το βασικό παράγοντα πλειοδυναμίας, Oct4, καθώς και στοιχεία του BMP σηματοδοτικού μονοπατιού, Smad5 and Id2, καθιστώντας το ως ρυθμιστή της διαφοροποίησης των ΕΒΚ. Τα αποτελέσματα αυτά υποδεικνύουν ένα νέο μηχανισμό αλληλεπίδρασης μεταξύ των παραπάνω μικρών-RNAs και των δύο κλάδων του TGF-β μονοπατιού, τονίζοντας τη σημαντικότητά τους σε αποφάσεις-κλειδιά για τη μοίρα των βλαστικών κυττάρων. Στο δεύτερο μέρος της ερευνητικής εργασίας, μελετήθηκε και συσχετίστηκε ο ρόλος της ΠΠΛ με την αδιαφοροποίητη κατάσταση των ΕΒΚ. Συγκεκριμένα, η αποσιώπηση της ΠΠΛ οδηγεί στην επαγωγή παραγόντων διαφοροποίησης των τριών βλαστικών στιβάδων και ενισχύει τη μεσοδερμική κατεύθυνση, ενώ αντίθετα η υπερέκφρασή της παρεμποδίζει τη διαφοροποίηση και διατηρεί το φαινότυπο των ΕΒΚ. Με τη χρήση μικροσυστοιχιών έκφρασης σε ΕΒΚ παρουσία και απουσία της ΠΠΛ, βρέθηκαν εκτεταμένες αλλαγές στο πρότυπο γονιδιακής έκφρασης. Μια ομάδα αυτορυθμιζόμενων γονιδίων εμπλέκεται στη μετάβαση από την αρχέγονη πλειοδύναμη στην ενεργοποιημένη-όψιμη κατάσταση επιβλάστης, ενώ μια άλλη σχετίζεται με τον κυτταρικό κύκλο και τα σηματοδοτικά μονοπάτια που εμπλέκονται στα ΕΒΚ. Επιπρόσθετα, ο επαναπρογραμματισμός ινοβλαστών από έμβρυο ποντικού με αποσιωπημένη τη ΠΠΛ δεν ήτανε επιτυχής, επισημαίνοντας ότι η ΠΠΛ είναι αναγκαία για την ολοκλήρωση της διαδικασίας. Ο πιθανός προτεινόμενος μηχανισμός δράσης της ΠΠΛ στον επαναπρογραμματισμό βασίζεται στη ρύθμιση της επιθηλιακής-μεσεγχυματικής μετάβασης μέσω του TGF-β μονοπατιού. Τα στοιχεία αυτά αποκαλύπτουν ότι η ΠΠΛ διαδραματίζει καθοριστικό ρόλο τόσο στη πλειοδυναμία των ΕΒΚ όσο και στον επαναπρογραμματισμό σωματικών κυττάρων

MicroRNAs for Fine-Tuning of Mouse Embryonic Stem Cell Fate Decision through Regulation of TGF-β Signaling

Over the past years, microRNAs (miRNAs) have emerged as crucial factors that regulate self-renewal and differentiation of embryonic stem cells (ESCs). Although much is known about their role in maintaining ESC pluripotency, the mechanisms by which they affect cell fate decisions remain poorly understood. By performing deep sequencing to profile miRNA expression in mouse ESCs (mESCs) and differentiated embryoid bodies (EBs), we identified four differentially expressed miRNAs. Among them, miR-191 and miR-16-1 are highly expressed in ESCs and repress Smad2, the most essential mediator of Activin-Nodal signaling, resulting in the inhibition of mesendoderm formation. miR-23a, which is also down-regulated in the differentiated state, suppresses differentiation toward the endoderm and ectoderm lineages. We further identified miR-421 as a differentiation-associated regulator through the direct repression of the core pluripotency transcription factor Oct4 and the bone morphogenetic protein (BMP)-signaling components, Smad5 and Id2. Collectively, our findings uncover a regulatory network between the studied miRNAs and both branches of TGF-β/BMP-signaling pathways, revealing their importance for ESC lineage decisions

Knockdown of p68 does not alter β-Catenin mRNA or protein expression.

<p>mRNA expression levels of p68 <b>A.</b> and β-Catenin <b>B.</b> in control (NS) and p68 siRNA-transfected LNCaP cells (+/− R1881 10 nM, 16 hours). QPCR data was normalised to GAPDH levels and fold change calculated relative to control (NS) (-R1881) mRNA levels (set as 1). The independent sample <i>t</i> test was used to compare differences in expression levels and show significance. <b>C.</b> Cropped immuno-blot images of lysates from LNCaP cells treated with 10 nM R1881 (16 hours) and transfected with control (NS) and p68 siRNA. Blots probed sequentially with β-Catenin, p68 and α-tubulin antibodies.</p

Over-expression of p68 additively enhances activity of β-Catenin mediated AR transcription.

<p>COS-7 cells transiently transfected in triplicate with p(ARE)₃Luc reporter and PCMV-β-galactosidase plasmids together with mammalian expression vectors for AR, β-Catenin and p68 (+/−10 nM R1881). Luciferase activity was corrected for the corresponding β-galactosidase activity to give relative activity. The range of plasmid levels (+ and ++) corresponds to 50 and 100 ng respectively. Data shown relative to AR activity alone (−R1881) (set as 1), and representative of at least n = 3 luciferase assay experiments (+/− SE). The independent sample <i>t</i> test was used to compare differences in expression levels and show significance.</p

p68 interacts with RNAPII in PCa cells and occupies many regions of the <i>PSA</i> gene.

<p><b>A.</b> Cropped immuno-blot images of LNCaP nuclear lysates immunoprecipitated with RNAP II H5 (ser-2), RNAP II H14 (ser-5), RNAP II CTD mouse monoclonal and p68 antibody (+/− R1881 10 nM, 8 hours), probed sequentially with p68 and RNAP II antibody. Extract samples contain nuclear cell lysate and protein G sepharose with no antibody present. Control samples contain antibody and protein G sepharose in nuclear extraction buffer only. <b>B.</b> Recruitment of AR and <b>C.</b> p68 to regions of the <i>PSA</i> gene. LNCaP cells were treated with 10 nM R1881 and harvested at 0, 15, 30, 45, 90 & 120 minute time points. Samples were immunoprecipitated with AR<b>,</b> p68 or control IgG antibodies and recovered material processed by ChIP assay. QPCR data are representative of n = 3 independent ChIP assays normalised to input levels (+/− SE). (N.B. QPCR primers could not be optimised to all exonic and intronic regions of the <i>PSA</i> gene). The independent paired sample <i>t</i> test was used to compare enrichment in recruitment between different <i>PSA</i> regions and show significance. <b>D.</b> Diagram of <i>PSA</i> gene depicting exon/intron boundaries.</p

Depletion of p68 reduces AR and β-Catenin recruitment to promoter regions of androgen responsive genes.

<p>LNCaP cells transfected with p68 or control (NS) siRNA, treated with 10 nM R1881 for 90 minutes and immunoprecipitated with either AR <b>A. B. </b><b>C. & D.</b> or β-Catenin <b>E. F. G. & H.</b> antibodies (including a IgG control antibody). Recovered material was processed by ChIP assay and recruitment to <i>PSA</i> ARE I (A & E), ARE III (B & F), <i>KLK2</i> (C & G) & <i>TMPRSS2</i> (D & H) promoter regions assessed relative to 0 minute time point. p68 depletion in LNCaP cells showed reduced AR & β-Catenin recruitment after treatment with 10 nM R1881 for 90 minutes to all regions assessed compared to control (NS) siRNA cells. Results shown represent n = 3 independent experiments (+/− SD). The independent sample <i>t</i> test was used to compare differences in expression levels and show significance.</p

Localisation and interaction of p68 and β-Catenin in PCa cells.

<p><b>A.</b> Cropped immuno-blot images show cytoplasmic and nuclear LNCaP and LNCaP-AI PCa cell lysates (+/− R1881 10 nM, 8 hours), probed sequentially with β-Catenin, AR, p68, α-tubulin and TATA binding (TBP) antibodies. <b>B.</b> Interaction of ectopic p68 and β-Catenin in COS-7 cells. Whole cell lysates of COS-7 cells transfected with pcDNA₃-p68-myc and pCS³⁺-Myc₆-β-Catenin constructs (+/− R1881 10 nM, 8 hours), were immunoprecipitated with β-Catenin and p68 antibody respectively. Cropped immuno-blots were probed sequentially with β-Catenin, p68 and myc antibody.<b>C</b>. Interaction of endogenous p68 and β-Catenin in the nucleus of LNCaP and LNCaP-AI PCa cells in the presence and absence of androgens. Cropped immuno-blot images of LNCaP and LNCaP-AI nuclear lysates, immunoprecipitated with p68 antibody (+/− R1881 10 nM, 8 hours), and probed sequentially with β-Catenin and p68. Extract samples contain either whole cell or nuclear lysate and protein G sepharose with no antibody present. Control (Con) samples contain antibody and protein G sepharose in extraction buffer only.</p