Linking open chromatin to pluripotency in ES cells : the role of the chromatin remodeler Chd1 ; Relacionando cromatina aberta com pluripotência em células estaminais embrionárias : o papel do remodelador da cromatina Chd1

Tese de doutoramento em Bioquímica (Biologia Molecular) apresentada à Fac. de Ciências e Tecnologia da Univ. de Coimbra ; Embryonic Stem (ES) cells are the prototypical pluripotent stem cells that can self-renew indefinitely and give rise to all cell types of the body. Through a transcriptional profile using microarrays, we have discovered that the mouse embryonic germ line have high transcriptional similarities to pluripotent stem cells, including mouse ES cells. A group of about 220 genes are up-regulated in pluripotent stem cells when compared to differentiated cells. From this cluster (that we called the pluricluster) we decided to test the effect of down-regulation in ES cells self-renewal and pluripotency. We developed a lentiviral RNA-interference screen to test the role of 41 candidate genes (chosen amongst the pluricluster with a bias towards transcription factors, chromatin remodelers, DNA/RNA binding proteins, oncogenes and unknown genes). Our screen identified 18 genes with RNAi phenotype including Chd1, NFYa, NFYb and Sall4. The last three genes allowed me to follow further characterization of their role in ES cells and reprogramming, as a collaboration project. However, Chd1 was the only novel regulator that had both self-renewal defects and loss of Oct4 activity in this screen. Chd1 is a chromatin remodeler that has been shown to recognize H3K4me3 and is associated with transcriptional activation and splicing. Our work shows clear evidence that Chd1 correlates with H3K4me3 enrichment, which overlaps with RNA polymerase II localization. Downregulation of Chd1 using RNAi in mouse ES cells, leads to increased heterochromatin foci marked by H3K9me3 and HP1. Moreover, in the absence of Chd1, ES cells have self-renewal and pluripotency defects. Chd1-deficient cells are not able to form a typical endodermal layer upon embryoid body differentiation, and have high propensity for neural differentiation. Chd1 also plays a role in the generation of iPS cells by over-expression of the four factors Oct4, Sox2, ...