Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge. Factors have been isolated from cell extracts that stimulate early steps in chromatin assembly in vitro . One such factor, chromatin assembly factor-1 (CAF-1), facilitates nucleosome formation coupled to DNA synthesis. It is thought to participate in a marking system at the crossroads of DNA replication and repair to monitor genome integrity and to define particular epigenetic states. We have now identified a chromatin assembly pathway independent of DNA synthesis involving the HIRA protein. Notably, CAF-1 is part of the histone H3 complex, H3.1 complex (replicative form) (Groth et al., 2007a, 2007b) and HIRA of the H3.3 complex (replacement form) (Tagami et al., 2004; Nakatani et al., 2004; Quivy et al., 2008; Loyola et al., 2009). In addition, another histone chaperone, Asf1, has to be integrated in a network of interactions leading to nucleosome deposition. A novel chaperone for the centromeric histone variant CENPA has been identified (Dunleavy et al., 2009). A major goal in our laboratory is to integrate the function of these factors and histone variants in vivo during development. This is considered in connection with replication, repair and control of histone pools. We will discuss our recent findings on this topic and the interrelationships with other assembly factors. Groth, A., Rocha, W., Verreault, A., Almouzni, G., 2007a. Chromatin challenges during DNA replication and repair. Cell 128, 721–733. Groth, A., Corpet, A., Cook, A., Roche, D., Bartek, J., Lukas, J., Almouzni, G., 2007b. Regulation of replication fork progression through histone supply/demand. Science 318, 1928–1931. Quivy, J.P., Gerard, A., Cook, A.J.L., Roche, D., Mone, M., Almouzni, G., 2008. A functional HP1–CAF-1 interaction module necessary to replicate and propagate pericentric heterochromatin impacts on S-phase progression in mouse cells. Nat. Struct. Mol. Biol. 15, 972–979. Dunleavy, E.M., Roche, D., Tagami, H., Lacoste, N., Ray-Gallet, D., Nakamura, Y., Daigo, Y., Nakatani, Y., Almouzni G., 2009. HJURP, a key CENP-A-partner for maintenance and deposition of CENP-A at centromeres at late telophase/G1. Cell 137, 485–497. Loyola, A., Tagami, H., Bonaldi, T., Roche, D., Quivy, J.P., Imhof, A., Nakatani, Y., Dent, S.Y.R., Almouzni, G., 2009. The HP1α-CAF-1-SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin. EMBO Rep. 10, 769–775. De Koning, L., Savignoni, A., Boumendil, C., Rehman, H., Asselain, B., Sastre-Garau, X., Almouzni, G., 2009. Heterochromatin protein 1α: a hallmark of cell proliferation relevant to clinical oncology. EMBO Mol. Med. 1, 178–191