Laurie A. Boyer et al. Cell, Vol. 122, 947-956, September 23, 2005.

Presentazione sul tema: "Laurie A. Boyer et al. Cell, Vol. 122, 947-956, September 23, 2005."— Transcript della presentazione:

1 Laurie A. Boyer et al. Cell, Vol. 122, 947-956, September 23, 2005

2 Regolatori master Ruolo chiave in pluripotenza e autorinnovamento

3 1) Identificare i geni target dei 3 fattori trascrizionali 2) Caratterizzare il circuito regolatore della trascrizione nelle cellule staminali embrionali umane

4 Arricchimento DNA mediante immunoprecipitazione cromatina e Identificazione geni target (ChIP on chip) OCT4, SOX2 e NANOG co-occupano una quota considerevole di geni target espressi nelle ES cells Alcuni di questi geni sono ATTIVATI e altri INIBITI per garantire pluripotenza e autorinnovamento Autoregolazione e loop di feedback positivo regolano l’espressione di OCT4, SOX2 e NANOG

5 * 1) CROSSLINK * 2) SONICATE * 3) IMMUNOPRECIPITATE * 4) UNLINK PROTEIN and PURIFY DNA * 5) qPCR QUALITY CONTROL

6 * 6) LABELING * 7) MICROARRAY HYBRIDIZATION * 8) WASH and SCAN

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9 VetrinoChr.PositionChr.PositionN.Probes 115575122464623039961 21224694779310872626939909 33109290599514756419339937 45147665548710628088439935 57106395416101504419039925 610151195961112969725139905 711129802259149411950039930 81494140702174133517539938 91741603407203004290039940 102030054185Y5768554739930 FineInizio

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12 Examples of OCT4 bound regions. Plots display unprocessed ChIP-enrichment ratios for all probes within a genomic region. Genes are shown to scale below plots (exons and introns are represented by thick vertical and horizontal lines, respectively), and the genomic region represented is indicated beneath the plot. The transcription start site and transcript direction are denoted by arrows.

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14 * regulate the expression levels of other genes by various mechanisms * located on human chromosome 1p22 * it is transcribed as a non-coding primary miRNA transcript, which is then processed into precursor miRNA and finally into the mature and functional miRNA of 21 to 25 nucleotidestranscribedmiRNAnucleotides * the mature miRNA functions by binding to 3’ UTR of multiple target mRNAs. This binding in turn results in an inhibition of translation of the target protein or degradation of the target messenger RNA * miR-137 is embedded within a CpG islandCpG island * implicated to act as a tumor suppressor in several cancer types including colorectal cancer, squamous cell carcinoma and melanoma via cell cycle controltumor suppressorcolorectal cancersquamous cell carcinomamelanoma * frequently silenced by promoter hyper-methylation in many tumour types, including colorectal, gastric and squamous cell carcinoma of the head and neck * directly inhibits CDK6 (Cyclin-dependent kinase 6) expression and decreases the level of phosphorylated RB (retinoblastoma), a known CDK6 downstream target. This is proposed to be the mechanism whereby miR-137 induces differentiation and inhibits proliferation of adult mouse neural stem cells, oligodendroma-derived stem cells, as well as human glioblastoma multiforme-derived stem cells. In addition, miR-137 targets Mib1 (Mind Bomb-1), a ubiquitin ligase known to be important for neurogenesis and neurodevelopmentCDK6 * in mouse embryonic stem cells (ESCs), Jarid1b (a histone H3 Lysine 4 demethylase) has recently been shown to be another direct target of miR-137. Jarid1b is frequently expressed early in mouse embryonic development and is thought to maintain the expression of undifferentiated ESC markersJarid1b miR-137 microRNA secondary structure and sequence conservation

15 * Pluripotenza vs. * Differenziamento

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17 OCT4, SOX2 and NANOG Co-occupy Each of Their Promoters

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19 * Progressi nell’abilità di coltivare e manipolare geneticamente cellule staminali embrionali umane * CONSENTONO DI  testare e manipolare il circuito regolatore della trascrizione nelle cellule staminali embrionali  produrre una mappa più completa del circuito  capire come queste cellule pluripotenti possono essere stimolate al differenziamento  capire come cellule differenziate possono essere riprogrammate verso uno stato pluripotente  in generale capire biologia cellulare, sviluppo, medicina rigenerativa

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