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Naja Vergani Triagem Funcional De Genes Envolvidos No Naja Vergani Triagem funcional de genes envolvidos no processo de manutenção da inativação do cromossomo X em humanos Functional screening of genes involved in the maintenance of X chromosome inactivation in humans São Paulo 2014 Naja Vergani Triagem funcional de genes envolvidos no processo de manutenção da inativação do cromossomo X em humanos Functional screening of genes involved in the maintenance of X chromosome inactivation in humans Tese apresentada ao Instituto de Biociências da Universidade de São Paulo, para a obtenção de Título de Doutor em Biologia, na Área de Genética. Orientadora: Profa. Dra. Lygia da Veiga Pereira Carramaschi São Paulo 2014 i Vergani, Naja Triagem funcional de genes envolvidos no processo de manutenção da inativação do cromossomo X em humanos. Número de páginas 241p. Tese (Doutorado) - Instituto de Biociências da Universidade de São Paulo. Departamento de Genética e Biologia Evolutiva. 1. Inativação do cromossomo X, 2. Triagem genômica funcional, 3. Epigenética, I. Universidade de São Paulo. Instituto de Biociências. Departamento de Genética e Biologia Evolutiva. Comissão Julgadora: ________________________ _____ _______________________ Prof(a). Dr(a). Prof(a). Dr(a). _________________________ ____________________________ Prof(a). Dr(a). Prof(a). Dr(a). Profa. Dra. Lygia da Veiga Pereira Carramaschi Orientadora ii Este trabalho foi realizado com auxílio financeiro das agências de fomento Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – PROEX – Projeto 33002010021P7), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). iii Dedicatória Para você meu vozinho querido Syllas Ermel, por todo imenso carinho, pela maravilhosa ajuda sempre e pelos lindos exemplos de vida. Vou carregá-los sempre comigo. E também para minha mãe Vivian , meu irmão Guido, minha vó Glacy e meu querido Thomas. Vocês são parte de mim. Amo muito vocês. iv Epígrafe “IF you can keep your head when all about you Are losing theirs and blaming it on you, If you can trust yourself when all men doubt you, But make allowance for their doubting too; If you can wait and not be tired by waiting, Or being lied about, don't deal in lies, Or being hated, don't give way to hating, And yet don't look too good, nor talk too wise; If you can dream - and not make dreams your master; If you can think - and not make thoughts your aim; If you can meet with Triumph and Disaster And treat those two impostors just the same; If you can bear to hear the truth you've spoken Twisted by knaves to make a trap for fools, Or watch the things you gave your life to, broken, And stoop and build 'em up with worn-out tools; If you can make one heap of all your winnings And risk it on one turn of pitch-and-toss, And lose, and start again at your beginnings And never breathe a word about your loss; If you can force your heart and nerve and sinew To serve your turn long after they are gone, And so hold on when there is nothing in you Except the Will which says to them: 'Hold on!'; If you can talk with crowds and keep your virtue, Or walk with Kings - nor lose the common touch, If neither foes nor loving friends can hurt you, If all men count with you, but none too much; If you can fill the unforgiving minute With sixty seconds' worth of distance run, Yours is the Earth and everything that's in it, And - which is more - you'll be a Man, my son!” Rudyard Kipling, If. v Agradecimentos À Profa. Dra. Lygia da Veiga Pereira, pela grande oportunidade, por todo o carinho, compreensão e apoio durante todos esses anos de trabalho. À Profa. Dra. Tatiana Teixeira Torres pela imensa ajuda, boa vontade e toda a paciência em me ensinar. À Profa. Dra. Jeanne Loring, pela acolhida e colaboração. À Mara, Profa. Dra. Ângela Maria Vianna Morgante, aos colegas Rob, Rathi e à Profa. Dra. Louise Laurent pela importante ajuda recebida nesse trabalho. Aos amigos de laboratório e do coração Simone, Ana Maria, Fabiano e Gustavo pela grande e constante ajuda, pela amizade de todo dia, pelos momentos de diversão e pelo carinho de vocês. E também à Érica e Joana pela super ajuda e à Fernanda, Luis, Max, Cynthia, Tiago, Bruno, Juliana, Giovana, Karla por todos os bons momentos compartilhados. À todos os amigos do departamento, entre eles Maraísa, Deisy, Helenice, Susy, Paulo, Lilian, Marianne, Marcos e Estela que muito me ajudaram. Especialmente à Fátima, pelas deliciosas conversas, pelo carinho e por todo o apoio. Aos Professores Regina Mingroni, Luciana Haddad e Luís Netto. Ao grande amigo Edmar Zanoteli por todas as conversas, apoio e incentivo. Um agradecimento especial ao meu querido “bomdrasto” Edmundo Roberto Doura (Lê) por todo imenso apoio, pelas risadas e por todo carinho. A Deus por colocar em meu caminho pessoas tão maravilhosas. vi Índice Dedicatória................................................................................................................iv Epígrafe.......................................................................................................................v Agradecimentos.........................................................................................................vi Lista de abreviaturas..................................................................................................xi Lista de figuras..........................................................................................................xiii Lista de tabelas........................................................................................................xvi 1. Introdução......................................................................................................1 1.1. Estrutura da cromatina e mecanismos de regulação epigenética...............2 1.1.1. Variantes de histonas...................................................................4 1.1.2. Modificações pós-traducionais em histonas..................................7 1.1.3. Modificações epigenéticas no DNA.............................................15 1.1.4. Papel dos RNAs não-codificadores.............................................18 1.2. Inativação do cromossomo X em mamíferos............................................23 1.3. Tipos de XCI..........................................................................................25 1.4. Genes que escapam à XCI......................................................................25 1.5. O centro de inativação do cromossomo X................................................27 1.6. Etapas da XCI........................................................................................30 1.6.1. Contagem do número de cromossomos X.................................30 1.6.2. Escolha dos futuros Xa e Xi......................................................32 1.6.3. Silenciamento do cromossomo X..............................................33 1.6.4. Manutenção do estado de inativação.........................................36 1.7. Uso de células HPRT+/- para o estudo da manutenção da XCI..................39 1.8. Uso de Bibliotecas de shRNA para Triagem Genômica Funcional............42 2. Objetivos.......................................................................................................46 3. Material e Métodos.........................................................................................49 3.1. Laboratórios e financiamentos..................................................................50 3.2. Linhagens celulares..................................................................................50 3.3. Cultivo de fibroblastos...............................................................................52 3.4. Passagem de fibroblastos.........................................................................53 vii 3.5. Congelamento de fibroblastos..................................................................53 3.6. Descongelamento de fibroblastos.............................................................54 3.7. Análise da eficiência de transdução viral em linhagens primárias de fibroblastos dérmicos humanos heterozigotas para o gene HPRT.............55 3.8. Extração de DNA de fibroblastos humanos primários................................57 3.9. Estração de RNA de fibroblastos humanos primários................................57 3.10. Produção de cDNA a partir de RNA...........................................................58 3.11. Quantificação de ácidos nucléicos para sequenciamento direto................58 3.12. Genotipagem de SNPs por sequenciamento direto....................................58 3.13. Teste de sensibilidade de fibroblastos humanos primários à droga 6- tioguanina...............................................................................................65 3.14. Seleção de fibroblastos humanos primários HPRT+ e HPRT-....................65 3.15. Cariotipagem de células da linhagem GM1661 selecionadas em meio 6TG............................................................................................................67 3.16. Geração e análise de curvas de crescimento celular.................................69 3.17. Escolha da biblioteca de shRNAs utilizada.................................................72 3.18. Otimização da transdução de fibroblastos primários humanos da linhagem GM1661 com partículas lentivirais copGFP................................................74 3.19. Transdução de fibroblastos primários humanos com a biblioteca de shRNAs......................................................................................................77
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