Senescence was initially described as a stable cell proliferation arrest resulting from the progression of primary human fibroblasts through a finite number of population doublings in vitro (35). However, activated oncogenes, oxidative stress, DNA damage, and drug-like inhibitors of specific enzymatic activities also induce senescence (14, 37, 82). In addition, senescence occurs in other cell types, such as primary human epithelial cells. In vivo, senescence is an important tumor suppression mechanism that restrains the proliferation of cells that harbor activated oncogenes (12, 16, 17, 51). Also, by limiting the self-renewal capacity of adult tissue stem cells, senescence is thought to contribute to tissue aging of many multicellular adult animals (38, 42, 53). Senescent cells are typically characterized by a large flat morphology and the expression of a senescence-associated β-galactosidase (SA β-gal) activity of unknown function (16, 21). In the nucleus of senescent cells, the chromatin undergoes dramatic remodeling through the formation of domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) (56, 57, 86). Cytologically, SAHF appear as compacted punctate DAPI (4,6-diamidino-2-phenylindole)-stained foci of DNA in senescent cell nuclei. The formation of SAHF is also reflected in a general increase in the resistance of nuclear chromatin to digestion by nucleases (57). SAHF contain modifications and associated proteins characteristic of transcriptionally silent heterochromatin, such as methylated lysine 9 of histone H3 (H3K9Me), heterochromatin protein 1 (HP1), and the histone H2A variant macroH2A. In addition, Narita et al. recently showed that high-mobility group A (HMGA) proteins, a family of abundant non-histone chromatin proteins, are essential structural components of SAHF (56). Proliferation-promoting genes, such as E2F target genes (e.g., cyclin A), are recruited into SAHF, dependent on the pRB tumor suppressor protein, thereby irreversibly silencing expression of those genes. Recently, we showed that two chromatin regulators, histone repressor A (HIRA) and antisilencing function 1a (ASF1a), drive the formation of SAHF in human cells (86). HIRA and ASF1a are the human orthologs of proteins known to create transcriptionally silent heterochromatin in yeasts, flies, and plants (9, 29, 39, 54, 63, 70-73, 78). In Saccharomyces cerevisiae, Hir1 and Hir2 are required for heterochromatin-mediated silencing of histone genes, telomeres, and mating loci, and the formation of pericentromeric chromatin structure (39, 70-73). Likewise, yeast Asf1p is required for heterochromatin-mediated silencing of telomeres, mating loci, and histone genes (40, 50, 70, 73, 75, 78) but also mediates nucleosome disassembly (2, 3, 68). Both HIRA and ASF1a bind to histones and exhibit histone chaperone activity in vitro (28, 64, 70, 78, 79). The HIRA/ASF1a-containing complex preferentially deposits the histone variant histone H3.3 into nucleosomes (46, 65, 76). Canonical human histone H3.1 and histone H3.3 differ in their primary amino acid sequences by only five amino acids. However, histone H3.1 is expressed periodically in the S phase of the cell cycle and is incorporated into chromatin during replication-coupled chromatin assembly (5, 36, 76). In contrast, histone H3.3 is expressed throughout the cell cycle and is incorporated into chromatin by the HIRA/ASF1a complex in a DNA replication- and repair-independent manner (5, 36, 76). Consistent with their partially overlapping biological and biochemical properties, yeast Asf1p and Hir proteins physically interact, and this interaction is necessary for telomeric silencing (19, 70). Likewise, the formation of SAHF in human cells by HIRA and ASF1a depends upon a physical interaction between these two proteins (76, 77, 86). A previous careful kinetic analysis of SAHF formation from our laboratory indicated that formation of SAHF is likely a multistep process (87). In the earliest defined step, the histone chaperone proteins HIRA and HP1 are both recruited to a specific subnuclear organelle, the acute promyelocytic leukemia (PML) nuclear body (10, 67). Most human cells contain 20 to 30 PML nuclear bodies, which are typically 0.1 to 1 μm in diameter and are enriched in the protein PML, as well as many other nuclear regulatory proteins (10, 67). PML bodies have been previously implicated in various cellular processes, including tumor suppression and cellular senescence (20, 23, 61). At a molecular level, they have been proposed as sites of assembly of macromolecular regulatory complexes and protein modification (24, 31, 61). After HIRA's translocation into PML bodies, chromatin condensation occurs, as defined by the appearance of DAPI-stained foci. Finally, H3K9Me accumulates, and HP1 and macroH2A proteins are recruited to SAHF. In this study, we set out to understand the series of events that contribute to the formation of SAHF in more detail and, in particular, to identify the molecular requirements for the different steps that were previously temporally defined. Here we report that during SAHF formation, each chromosome condenses into a single DAPI focus. Chromosome condensation mediated by the histone chaperone ASF1a depends on its binding to histone H3, as well as HIRA. Interestingly, HP1γ, but not HP1α and HP1β, is phosphorylated on serine 93 in senescent cells. This phosphorylation is not required for the protein's localization to PML bodies, but is required for its binding to SAHF. Remarkably, a large reduction in the amount of chromatin-bound HP1 proteins does not affect chromosome condensation, recruitment of histone variant macroH2A to SAHF, expression of SA β-gal, or senescence-associated cell cycle exit. Based on these data, we propose a multistep model of dependent and independent steps that culminate in the formation of mature SAHF.