Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.

Distinct patterns of posttranslational histone modifications can regulate DNA-templated events such as mitosis, transcription, replication, apoptosis, and DNA damage, suggesting the presence of a "histone code" in these nuclear processes. Phosphorylation of histone H2A S129 at sites of DNA double-strand breaks (DSBs) has been implicated in damage repair in yeast. Here, we describe another phosphorylation event on serine 1 (S1) of histone H4; this event is also associated with MMS- or phleomycin-induced DSBs but not with UV-induced DNA damage. Chromatin-immunoprecipitation (ChIP) studies of an HO-endonuclease-inducible strain show that S1 phosphorylation is specifically enhanced 20- to 25-fold in nucleosomes proximal to the DSB. In addition, we show that casein kinase II (CK2) can phosphorylate H4 S1 in vitro and that null or temperature-sensitive CK2 yeast mutants are defective for induction of H4 S1 phosphorylation upon DNA damage in vivo. Furthermore, H4 S1 phosphorylation and CK2 play a role in DSB re-joining as indicated by a nonhomologous end-joining (NHEJ) plasmid assay. CK2 has been implicated in regulating a DNA-damage response; our data suggest that histone H4 S1 is one of its physiological substrates. These data suggest that this modification is a part of the DNA-repair histone code.

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.

Folylpoly-␥-glutamate synthetase (FPGS) catalyzes the activation of folate antimetabolites in mammalian tissues and tumors. We have determined the sequence, abundance, and function of human FPGS transcripts and found some striking differences to transcription of the mouse gene that allow production of FPGS isoforms in mouse liver and dividing tissues. Multiple human transcripts were identified, including the homolog of the mouse transcripts that initiate at two upstream exons. However, the human FPGS upstream promoter is infrequently used, and transcripts from this promoter include sequences homologous with only one of the upstream exons found in the mouse. The downstream promoter generates an array of transcripts, some of which do not produce active enzyme, a phenomenon not seen in the mouse. Hence, the dual promoter mechanism directing expression of FPGS isozymes in mouse tissues is not conserved in humans, and, unlike the mouse downstream promoter, the human downstream promoter is active in both dividing and differentiated tissues. This study raises questions about the differences in function served by the two mouse FPGS isozymes and how, or if, human tissues fulfill these functions. How humans and mice produce FPGS in only a subset of tissues using such different promoter structures also becomes a central issue.

The mouse fpgs gene uses two distantly placed promoters to produce functionally distinct isozymes in a tissue-specific pattern. We queried how the P1 and P2 promoters were differentially controlled. DNA methylation of the CpG-sparse P1 promoter occurred only in tissues not initiating transcription at this site. The P2 promoter, which was embedded in a CpG island, appeared open to transcription in all tissues by several criteria, including lack of DNA methylation, yet was used only in dividing tissues. The patterns of histone modifications over the two promoters were very different: over P1, histone activation marks (acetylated histones H3 and H4 and H3 trimethylated at K4) reflected transcriptional activity and apparently reinforced the effects of hypomethylated CpGs; over P2, these marks were present in tissues whether P2 was active, inactive, or engaged in assembly of futile initiation complexes. Since P1 transcriptional activity coexisted with silencing of P2, we sought the mechanism of this transcriptional interference. We found RNA polymerase II, phosphorylated in a pattern consistent with transcriptional elongation, and only minimal levels of initiation factors over P2 in liver. We concluded that mouse fpgs uses DNA methylation to control tissue-specific expression from a CpG-sparse promoter, which is dominant over a downstream promoter masked by promoter occlusion.

In this postgenome era, understanding how a cell regulates access to information encoded in the deoxyribonucleic acid (DNA) is essential. In eukaryotic cells, DNA is bound to histone proteins to form chromatin fibers. Numerous studies have now shown that post-translational histone modifications play an important role in regulating the access of DNA-dependent proteins to the DNA template. Determining the status of histone modifications in a genomic region has proven to yield information on the chromatin structure and the regulation of a specific gene in vivo. Chromatin immunoprecipitation (ChIP) allows researchers to determine the status of both histone modifications and the nuclear effector proteins located at gene of interest. ChIP, if applied globally, can also reveal how chromatin structures are dynamically changed when cells respond to certain stimuli. In this chapter, we describe this powerful technique in detail.