Next, we wondered whether a similar requirement for CAF-1 could also be observed in ES cells, which are derived from the blastocyst inner cell mass. Given the early developmental arrest observed in Chaf1a −/− embryos, such cells could not be derived directly from null embryos, and we thus used an RNAi strategy (Figure 2A). p150CAF-1 knockdown in ES cells was quantified by Western blot analysis and IF. (Figure S1). p150CAF-1 RNAi-depleted ES cells displayed a phenotype very similar to the cells of Chaf1a −/− E4 mice. DAPI-dense foci were lost, and we observed diffuse HP1α and DAPI staining around the nucleoli and at the periphery of the nucleus (Figure 2B). This result indicates that p150CAF-1 is essential for nuclear organization of heterochromatin in ES cells in a similar way to early pre-implantation embryos. Surprisingly, we did not observe this severe alteration in heterochromatin organization in primary mouse embryonic fibroblasts (MEFs) following p150CAF-1 depletion by RNAi. p150CAF-1 depletion in MEFs resulted in a strong inhibition of cell proliferation (unpublished data), but did not alter the clustering of heterochromatin domains (Figures 2C and S2). Similarly, p150CAF-1 depletion in 3T3 cells did not affect heterochromatin organization [16]. These observations reveal a specific function for CAF-1 in the early embryo and ES cells.