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The Repression of MEF2 Transcription Factors Exerted by Class Iia Hdacs

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The Repression of MEF2 Transcription Factors Exerted by Class Iia Hdacs 81,9(56,7<2)8',1( BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB 3K'Course in Biomedical Sciences and Biotechnology ;;9,,&<&/( THE REPRESSION OF MEF2 TRANSCRIPTION FACTORS EXERTED BY CLASS IIA HDACS AND THEIR DEGRADATION STIMULATED BY CDK4 DETERMINE THE ACQUISITION OF HALLMARKS OF TRANSFORMATION IN FIBROBLASTS. 3K'6WXGHQW'L*LRUJLR(URV 7XWRUSURI&ODXGLR%UDQFROLQL (URV'L*LRUJLR To my family, Sara and those who believe in the research against cancer ABSTRACT 1 RIASSUNTO 2 INTRODUCTION 3 1. The HDACs world 3 2. Class IIa HDACs: similarities and differences between class IIa and class I HDACs 4 3. Class IIa HDACs: HDACs with orphan substrates or missed during evolution? 7 4. Pathways of regulation 10 a) regulation of class IIa HDACs transcription and modulation of the stability of the messengers (RNAi) 10 b) sub-cellular localization 11 5. Partners and biological functions 20 6. Class IIa HDACs as regulators of proliferation and cancer 28 7. MEF2 family of transcription factors 37 8. On the molecular basis of the MEF2-Class IIa HDACs axis: structure of MEF2/DNA, MEF2/Cabin1/DNA, MEF2/HDAC9/DNA and MEF2/DNA/p300 complexes. 38 9. Pathways of regulation 40 a) binding to repressors and co-activators 41 b) post-translational modifications 42 c) regulation of MEF2s transcription and modulation of the stability of the messengers (RNAi) 49 d) regulation of protein stability 51 10. Main functional roles 53 11. MEF2 as a regulator of differentiation programs 54 12. A lesson from the study of the knock-outs. 56 The role of MEF2s and of the MEF2-class IIa HDACs axis in myogenesis 57 The role of MEF2 TFs in cardiomyogenesis 59 The role of MEF2 TFs and of the MEF2-class IIa HDACs axis in endochondral bone ossification 59 The role of MEF2s and of the MEF2-class IIa HDACs axis in vasculogenesis and differentiation of vascular smooth muscle cells 60 The role of MEF2 TFs in neuronal development 60 The role of MEF2s in hematopoiesis and T cell development 61 The role of MEF2s in melanogenesis 62 The role of MEF2s in neural crest development 62 1. MEF2A ais roles in ell-differentiated adult tissues: evidences of regulation of hypertrophy, metabolism and synaptic plasticity 62 14. MEF2s as regulators of proliferation and cancer 64 MEF2 and NOTCH1 73 AIMS 75 MATERIALS AND METHODS 77 RESULTS 83 The forced activation of HDAC4 triggers morphological changes in murine NIH-3T3 fibroblasts and increases their proliferation rate. 83 HDAC4/TM over-expressing cells acƋuire ͞classical͟ hallmarŬs of transformation. 85 Identification of genes under the influence of HDAC4. 87 The oncogenic activity of HDAC4/TM is largely dependent on MEF2 repression. 87 Several genes repressed by HDAC4 are MEF2 targets and are negatively regulated by the PI3K/Akt pathway 89 The PI3K/Akt pathway represses MEF2 transcriptional activity. 91 Oncogene dependent regulation of MEF2 activities. 94 MEF2C and MEF2D protein stability is regulated during the cell-cycle. 96 Skp2 regulates MEF2C and MEF2D stability. 97 MEF2 N-terminus domain is involved in the interaction with SKP2. 101 MEF2D is a substrate of CDK4. 103 Roles of MEF2s in the regulation of cell-cycle progression. 104 CDKN1A is a key element in the anti-proliferative activity of MEF2. 107 MEF2 TFs bind the first intron of p21 CDKN1A and promote the acetylation of H3K27, thus enhancing p21 transcription 107 The relevance of MEF2-class IIa HDACs axis perturbation in tumours. 110 DISCUSSION 115 ACKNOWLEDGEMENTS 119 REFERENCES 121 APPENDIX 151 Abstract BSTCT The repression of MEF2 transcription factors exerted by class IIa HDACs and their degradation stimulated by CDK4 determine the acquisition of hallmarks of transformation in fibroblasts. MEF2 transcription factors (TFs) are well known regulators of differenziative and adaptive responses, with predominant roles in muscular, cerebral and immune districts. However, literature concerning the contribution of MEF2 TFs in processes of transformation and oncogenesis is scattered and contradictory; class IIa HDACs (HDAC4, HDAC5, HDAC7, HDAC9) are well-established repressors of MEF2 activity and increasing numbers of selective class IIa HDACs inhibitors are under preclinical screening for various diseases, including cancer. However, a clear demonstration of the oncogenic functions of these proteins is still missing. The aim of this work was to clarify the possible involvement of the HDAC-MEF2 axis in carcinogenesis using as a model different mesenchymal cell lines with varying degrees of immortalization. Here, we incontrovertibly demonstrate a pro-oncogenic role of a nuclear resident form of HDAC4/HDAC7 in NIH-3T3 and BALB/c fibroblasts. Through a DNA microarray experiment we identified the signature of HDAC4 and, as expected, among the genes directly repressed by HDAC4 many are MEF2 targets. We demonstrated that most of the transforming potential of HDAC4 is due to the repression of MEF2 transcriptional activity and that the MEF2- HDAC axis is particularly active in Soft-tissue Sarcomas; in these tumors the binding between HDAC4 and MEF2 could be an effective therapeutic target, as proved by us in vitro. We also demonstrated that the repression of MEF2 activity could also be exerted by common oncogenes, such as RAS and AKT, which act independently from class IIa HDACs by inducing a decrease in the half-life of MEF2C and MEF2D proteins. We reported that MEF2C/D are subjected to a cyclic degradation during cell-cycle with peaks of dysregulation concomitant with S phase entry. The signal that controls the cyclic degradation of MEF2 is the phosphorylation by CDK4/CyclinD1 on two serine residues, conserved among the MEF2 family members, except for MEF2B and a transcriptional variant expressed in skeletal muscles. As a consequence of this phosphorylation, MEF2C/D are bound by the E3-ligase SKP2 that mediates their poly-ubiquitylation and degradation in the proteasome. The cyclic degradation of MEF2 proteins is required for the correct progression of the cell-cycle, as any interference in this degradation process causes an arrest in G1 because of MEF2- mediated transcription of p21/CDKN1A; on the contrary, any increase in MEF2 degradation causes an aberrant progression in the cell-cycle, a common feature of cancer cells. In summary, we demonstrated that in fibroblasts MEF2 activity could be alternatively repressed by class IIa HDACs or through a cell-cycle based degradation process; in both the cases MEF2 repression results in an increase in cell proliferation and in the acquisition of hallmarks of transformation. 1 Riassunto SST La repressione dei fattori di trascrizione MEF2 esercitata dalle Iston Deacetilasi di classe IIa e la loro degradazione stimolata da CDK4 determina l'acquisizione di caratteristiche maligne nei fibroblasti. I fattori di trascrizione MEF2 sono regolatori ben noti di risposte differenziative e di adattamento, con ruoli predominanti nei distretti muscolari, cerebrali e del sistema immunitario. Tuttavia, la letteratura riguardante il contributo dei fattori MEF2 nei processi di trasformazione e di oncogenesi è contraddittoria; le Iston Deacetilasi di classe IIa (HDAC4, HDAC5, HDAC7, HDAC9) sono forti repressori dell attività dei fattori MEF2 e un numero crescente di inibitori selettivi di queste proteine stanno entrando in fase clinica per lapplicazione sperimentale in diverse patologie, compreso il cancro. Tuttavia, una chiara dimostrazione delle funzioni oncogeniche di queste proteine è ancora mancante. Lo scopo di questo lavoro è stato quello di chiarire il possibile coinvolgimento dell'asse HDAC4/7-MEF2 nella carcinogenesi utilizzando come modello diverse linee di cellule mesenchimali a vari gradi di immortalizzazione. Qui, dimostriamo incontrovertibilmente un ruolo pro- oncogenico di una forma residente nucleare di HDAC4 e HDAC7 in fibroblasti murini NIH-3T3 e BALB/c. Mediante un esperimento di DNA microarray abbiamo identificato la firma genica di HDAC4 e, come previsto, tra i geni repressi direttamente da HDAC4 molti sono regolati da MEF2. Abbiamo dimostrato che la maggior parte del potenziale trasformante di HDAC4 è dovuta alla repressione dellattività trascrizionale di MEF2 e che lasse HDAC4/7-MEF2 è particolarmente attivo nei sarcomi dei tessuti molli; in questi tumori il legame tra HDAC4 e MEF2 potrebbe essere un bersaglio terapeutico efficace, come dimostrato da noi in vitro. Abbiamo anche dimostrato che oncogeni comuni, quali RAS e AKT, reprimono lattività di MEF2 agendo in un modo indipendente dalle HDAC di classe IIa e destabilizzando a livello proteico MEF2C e MEF2D. In particolare, MEF2C/D sono sottoposti ad una degradazione ciclica ad ogni ciclo cellulare con punte di disregolazione in concomitanza allingresso in fase S. Il segnale che controlla la degradazione ciclica dei MEF2 è la fosforilazione da parte di CDK4/CyclinD1 su due residui conservati tra i membri della famiglia MEF2, tranne che da MEF2B e da una variante trascrizionale diffusa nei muscoli scheletrici. Come conseguenza di questa fosforilazione, MEF2C/D vengono legati dallE3 ligasi SKP2 che media la loro poli-ubiquitinazione e degradazione nel proteasoma. La degradazione ciclica delle proteine MEF2 è necessaria per la corretta progressione del ciclo cellulare e qualsiasi interferenza in questo processo di degradazione altamente regolato provoca un arresto in G1 a causa dellinduzione da parte di MEF2 del regolatore negativo del ciclo p21/CDKN1A; al contrario, un aumento della degradazione di MEF2 provoca una progressione aberrante nel ciclo cellulare, una caratteristica
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