Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G₂ phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G₂/M transition. During the G₂ phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.

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.

The mammalian circadian clock resides in neurons of the hypothalamic suprachiasmatic nucleus (SCN). Light entrains phase resetting of the clock using the retino-hypothalamic tract, via release of glutamate. Nighttime light exposure causes rapid, transient induction of clock and immediate–early genes implicated in phase-shifting the pacemaker. Here we show that a nighttime light pulse caused phosphorylation of Ser10 in histone H3's tail, in SCN clock cells. The effect of light was specific, and the kinetics of H3 phosphorylation were characteristic of the early response, paralleling c-fos and Per1 induction. Using fos-lacZ transgenic mice, we found that H3 phosphorylation and Fos induction occurRed in the same SCN neurons. Systemic treatment with the GABA_B receptor agonist baclofen prevented light-induced c-fos and Per1 expression and H3 phosphorylation, indicating that one signaling pathway governs both events. Our results suggest that dynamic chromatin remodeling in the SCN occurs in response to a physiological stimulus in vivo.

Plasticity in gene expression is achieved by a complex array of molecular mechanisms by which intracellular signaling pathways directly govern transcriptional regulation. In addition to the remarkable variety of transcription factors and co-regulators, and their combinatorial interaction at specific promoter loci, the role of chromatin remodeling has been increasingly appreciated. The N-terminal tails of histones, the building blocks of nucleosomes, contain conserved residues that can be post-translationally modified by phosphorylation, acetylation, methylation and other modifications. Depending on their nature, these modifications have been linked to activation or silencing of gene expression. We wanted to investigate whether neuronal stimulation by various signaling pathways elicits chromatin modifications that would allow transcriptional activation of immediate early response genes. We have analysed the capacity of three drugs – SKF82958 (a dopaminergic receptor agonist), pilocarpine (a muscarinic acetylcholine receptor agonist) and kainic acid (a kainate glutamate receptor agonist) – to induce chromatin remodeling in hippocampal neurons. We show that all stimulations induce rapid, transient phosphorylation of histone H3 at serine 10. Importantly, the same agonists induce rapid activation of the mitogen-activated protein kinase pathway with similar kinetics to extracellular-regulated-kinase phosphorylation. In the same neurons where this dynamic signaling cascade is activated, there is induction of c-fos transcription. Histone H3 Ser10 phosphorylation is coupled to acetylation at the nearby Lys14 residue, an event that has been linked to local opening of chromatin structure. Our results underscore the importance of dynamic chromatin remodeling in the transcriptional response to various stimuli in neuronal cells.

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die.

The Aurora kinases are cell cycle-regulatory serine-threonine kinases that have been implicated in the function of the centrosomes, kinetechores, chromosome dynamics, and cytokinesis. In comparison with other tissues, there are high levels of expression of Aurora-B and -C in testis. What their respective roles in mammalian spermatogenesis are is an open question. Here we describe the expression and distribution patterns of the three kinases in mouse testis using in situ hybridization and immunohistochemistry. Importantly, the localization of Aurora-B is tightly regulated during spermatogenesis, whereas Aurora-C expression appears to be testis specific. To address the function of Aurora-B in spermatogenesis, we have generated transgenic mice using a pachytene-stage-specific promoter driving the expression of either wild-type Aurora-B or an inactive form of the kinase. Expression of the inactive Aurora-B results in abnormal spermatocytes, increased apoptosis, spermatogenic arrest, and subfertility defects. The function of Aurora-C may also be targeted in the Aurora-B transgenic mutants. To address the function of Aurora-C in testis, we generated Aurora-C knockout mice by homologous recombination. Remarkably, Aurora-C null mice were viable, yet the males had compromised fertility. Aurora-C mutant sperm display abnormalities that included heterogenous chromatin condensation, loose acrosomes, and blunted heads. These findings indicate that Aurora-B and Aurora-C serve specialized functions in mammalian spermatogenesis.