Résumé
Cell differentiation is a multi-step process marked by progressive silencing of gene expression through mechanisms believed to involve heterochromatin. We have previously shown that interaction between the Kr?l associated box-containing zinc finger proteins (KRAB-ZFP) corepressor TIF1beta and the heterochromatin proteins HP1 is essential for progression through differentiation of embryonal carcinoma F9 cells. This analysis clearly demonstrated the link between gene specific repressors, components of heterochromatin and cell differentiation. In mammals, there are three HP1 isotypes, HP1alpha, beta, and gamma, that appear to be involved in both eu- and heterochromatin, but whose individual functions are still poorly defined. Therefore, the aim of the present study was to determine in vivo (i) which HP1 isotypes interact with TIF1beta, (ii) in which sub-nuclear compartments these interactions occur and (iii) whether these interactions are regulated during cell differentiation. To address these questions, we established stable F9 cell lines co-expressing TIF1beta fused to the ECFP fluorophore and HP1alpha, beta, or gamma fused to the EYFP fluorophore. Using the F?r resonance energy transfer (FRET) technology, we map the physical interaction between TIF1beta-CFP and the different HP1-YFP isotypes in living F9 cells. We demonstrate that in non-differentiated cells, TIF1beta-CFP/HP1-YFP interaction occurs only within euchromatin and involves selectively HP1beta-YFP and HP1gamma-YFP, but not HP1alpha-YFP. Furthermore, in differentiated cells, TIF1beta-CFP selectively associates with HP1beta-YFP within heterochromatin, while TIF1beta-CFP/HP1gamma-YFP is exclusively present within euchromatin. No physical TIF1beta-CFP/HP1alpha-YFP interaction is detected in neither non differentiated nor differentiated cells. These results support the notion that, in vivo, HP1 isotypes have specific nonredundant functions and provide evidence for HP1beta playing an essential role in the shuttling of TIF1beta from eu- to heterochromatin during cell differentiation.