Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA.

In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.

Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity.

DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.

Biochemical analysis of the cell cycle and cell cycle checkpoints in transiently transfected cells after collection with magnetic beads.

It is frequently necessary to examine the biochemical effects of ectopically expressed proteins or short hairpin (sh) RNA-mediated protein knock-down in intact cells. Plasmids that direct the expression of ectopic proteins or shRNAs can be conveniently introduced into cells by transient transfection of plasmid DNAs. However, most protocols used for the transient transfection of plasmid DNAs introduce the foreign DNA into only a minority of the total cells. Therefore, to investigate the biochemical effects of the foreign DNA it is necessary to purify the transfected cells away from the untransfected cells. This can be easily achieved by cotransfection of a plasmid encoding the cell surface marker protein CD19 or CD20, followed by immunopurification of the CD19- or CD20-expressing cells with magnetic beads coated with an anti-CD19 or anti-CD20 antibody. The purified cells can be used for a wide range of biochemical analyses, including protein extraction for Western blot and immunoprecipitation, RNA extraction for Northern blot, and DNA and chromatin extraction for nuclease digestion. Since the CD19/CD20 cell surface marker approach can be readily combined with analysis of cell cycle distribution of propidium-iodide-stained cells, it is straightforward to simultaneously determine the biochemical and cell cycle effects of an ectopically expressed or knocked-down protein.

Defective S phase chromatin assembly causes DNA damage, activation of the S phase checkpoint, and S phase arrest.

The S phase checkpoint protects the genome from spontaneous damage during DNA replication, although the cause of damage has been unknown. We used a dominant-negative mutant of a subunit of CAF-I, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S phase chromatin assembly and found that this induced S phase arrest. Arrest was accompanied by DNA damage and S phase checkpoint activation and required ATR or ATM kinase activity. These results show that in human cells CAF-I activity is required for completion of S phase and that a defect in chromatin assembly can itself induce DNA damage. We propose that errors in chromatin assembly, occurring spontaneously or caused by genetic mutations or environmental agents, contribute to genome instability.