and Molecular DNA around a histone octamer core particle containing one H3-H4 tetramer and two H2A-H2B dimers. The valid-Genetics Box 800733 ity of this model has been confirmed by determining the crystal structures of the histone octamer, and of the Charlottesville, Virginia 22908 † Institut de Ge ´ne ´tique et de Biologie Mole ´culaire nucleosome particle (Arents et al., 1991; Richmond et al., 1993; Luger et al., 1997). As revealed by these and et Cellulaire CNRS-INSERM-ULP, B. P. 163 other studies, the C-terminal histone-fold domains of core histones have similar conformations that are critical 67404 Illkirch, Strasbourg France for the assembly of nucleosomes by mediating histonehistone and histone-DNA interactions (reviewed in Wolffe, 1998). In contrast, the N-terminal tails of core histones are less structured and are not essential for Signal Transduction and Chromatin maintaining the integrity of nucleosomes since removal The ability to detect extracellular stimuli and execute the of these tails by trypsin treatment does not diminish appropriate response is crucial to all cellular functions. nucleosome stability (Whitlock and Simpson, 1977; Au-Upon receiving external signals, be it growth factor stimsio et al., 1989). Instead, histone tails are thought to ulation or exposure to stress, distinct pathways are actimake secondary and more flexible contacts with DNA vated that culminate in the induction or repression of and adjacent nucleosomes (Luger et al., 1997) that allow defined sets of genes. The transduction of signals from for dynamic changes in the accessibility of the underlycell surface to the nucleus often involves phosphorylaing genome. tion cascades that not only allow for rapid transmission, How do these N-terminal histone tails participate in but also serve to amplify the signal by activating multiple the modulation of chromatin architecture? One possible factors and genes. The finely tuned combination of these way is that they constitute targets for ATP-dependent signal-induced nuclear effects thus leads to integrated chromatin remodeling factors such as Swi/Snf and cellular responses such as proliferation, differentiation, NURF (Georgel et al., 1997; Lee et al., 1999; Krebs et and apoptosis. al., 2000). Readers interested in the function of these A central goal in the signaling field is the identification remodeling complexes are directed to other reviews of physiologically relevant targets of transduction pathmore focused on that topic (Kingston and Narlikar, 1999; ways. To that end, much attention has been focused Peterson and Workman, 2000). Another way is that these on the signal-induced activation of transcription factors tails are subjected to a diverse array of posttranslational and components of the transcription machinery. Howmodifications, such as acetylation and phosphorylation ever, chromatin, the physiological packaging structure (Figure 1), which may modulate the contacts between of histones and genomic DNA, has largely been nehistones and DNA. Because these modifications are regl...

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. Here we report that these two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these dimodified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression.

The AMP-activated protein kinase (AMPK) cascade plays an important role in the regulation of energy homeostasis within the cell. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). We have isolated and characterized two isoforms of the gamma subunit, termed gamma2 and gamma3. Both gamma2 (569 amino acids) and gamma3 (492 amino acids) have a long N-terminal domain which is not present in the previously characterized isoform, gamma1. As with gamma1, mRNA encoding gamma2 is widely expressed in human tissues, whereas significant expression of gamma3 mRNA was only detected in skeletal muscle. Using isoform-specific antibodies, we determined the AMPK activity associated with the different gamma isoforms in a number of rat tissues. In most tissues examined more than 80% of total AMPK activity was associated with the gamma1 isoform, with the remaining activity being accounted for mainly by the gamma2 isoform. Exceptions to this were testis and, more notably, brain where all three isoforms contributed approximately equally to activity. There was no evidence for any selective association between the alpha1 and alpha2isoforms and the various gamma isoforms. However, the AMP-dependence of the kinase complex is markedly affected by the identity of the gamma isoform present, with gamma2-containing complexes having the greatest AMP-dependence, gamma3 the lowest, and gamma1 having an intermediate effect. Labelling studies, using the reactive AMP analogue 8-azido-[(32)P]AMP, indicate that the gamma subunit may participate directly in the binding of AMP within the complex.

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic "histone code."

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP-and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIFbinding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.

During the immediate-early response of mammalian cells to mitogens, histone H3 is rapidly and transiently phosphorylated by one or more unidentified kinases. Rsk-2, a member of the pp90rsk family of kinases implicated in growth control, was required for epidermal growth factor (EGF)-stimulated phosphorylation of H3. RSK-2 mutations in humans are linked to Coffin-Lowry syndrome (CLS). Fibroblasts derived from a CLS patient failed to exhibit EGF-stimulated phosphorylation of H3, although H3 was phosphorylated during mitosis. Introduction of the wild-type RSK-2 gene restored EGF-stimulated phosphorylation of H3 in CLS cells. In addition, disruption of the RSK-2 gene by homologous recombination in murine embryonic stem cells abolished EGF-stimulated phosphorylation of H3. H3 appears to be a direct or indirect target of Rsk-2, suggesting that chromatin remodeling might contribute to mitogen-activated protein kinase-regulated gene expression.

TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38a at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor-a (TNF-a), interleukin-1 (IL-1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre-incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the ®rst residue phosphorylated by SAPK2a/p38a that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS-stimulated macrophages, and by proin¯amma-tory cytokines or osmotic shock in epithelial KB cells or embryonic ®broblasts. The activation of TAK1 by TNF-a, IL-1 or osmotic shock was also enhanced in embryonic ®broblasts from SAPK2a/p38a-de®cient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38a-mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38a but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).

Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9). Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe. The chromodomain of mouse HP1 (and Swi6 in S. pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly. We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin. In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length. Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome. These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.