Interphase H1 phosphorylation: Regulation and functions in chromatin.

Many metazoan cell types differentially express multiple non-allelic amino acid sequence variants of histone H1. Although early work revealed that H1 variants, collectively, are phosphorylated during interphase and mitosis, differences between individual H1 variants in the sites they possess for mitotic and interphase phosphorylation have been elucidated only relatively recently. Here, we review current knowledge on the regulation and function of interphase H1 phosphorylation, with a particular emphasis on how differences in interphase phosphorylation among the H1 variants of mammalian cells may enable them to have differential effects on transcription and other chromatin processes.

H3 phosphorylation: dual role in mitosis and interphase.

Chromatin condensation and subsequent decondensation are processes required for proper execution of various cellular events. During mitosis, chromatin compaction is at its highest, whereas relaxation of chromatin is necessary for DNA replication, repair, recombination, and gene transcription. Since histone proteins are directly complexed with DNA in the form of a nucleosome, great emphasis is put on deciphering histone post-translational modifications that control the chromatin condensation state. Histone H3 phosphorylation is a mark present in mitosis, where chromatin condensation is necessary, and in transcriptional activation of genes, when chromatin needs to be decondensed. There are four characterized phospho residues within the H3 N-terminal tail during mitosis: Thr3, Ser10, Thr11, and Ser28. Interestingly, H3 phosphorylated at Ser10, Thr11, and Ser28 has been observed on genomic regions of transcriptionally active genes. Therefore, H3 phosphorylation is involved in processes requiring opposing chromatin states. The level of H3 phosphorylation is mediated by opposing actions of specific kinases and phosphatases during mitosis and gene transcription. The cellular contexts under which specific residues on H3 are phosphorylated in mitosis and interphase are known to some extent. However, the functional consequences of H3 phosphorylation are still unclear.

Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy.

In Mycobacterium tuberculosis, the sensor kinases DosT and DosS activate the transcriptional regulator DosR, resulting in the induction of the DosR regulon, which is important for anaerobic survival and perhaps latent infection. The individual and collective roles of these sensors have been postulated biochemically, but their roles in vivo have remained unclear. This work demonstrates distinct and additive roles for each sensor during anaerobic dormancy. Both sensors are necessary for wild-type levels of DosR regulon induction, and concomitantly, full induction of the regulon is required for wild-type anaerobic survival. In the anaerobic model, DosT plays an early role, responding to hypoxia. DosT then induces the regulon and with it DosS, which sustains and further induces the regulon. DosT then loses its functionality as oxygen becomes limited, and DosS alone maintains induction of the genes from that point forward. Thus, M. tuberculosis has evolved a system whereby it responds to hypoxic conditions in a stepwise fashion as it enters an anaerobic state.

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor.

A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe3+) form, whereas DosT exists in the O2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.

Mutational analysis of the Myxococcus xanthus Omega4406 promoter region reveals an upstream negative regulatory element that mediates C-signal dependence.

C signaling plays a key role in coordinating cell movement and differentiation during the multicellular developmental process of Myxococcus xanthus. C signaling regulates expression of genes induced after about 6 h into development, when cells are forming mounds. One gene whose expression depends absolutely on C signaling was identified by insertion of a transposable element at site Omega4406 which generated a transcriptional fusion between lacZ and an upstream promoter. We have investigated regulation of the Omega4406 promoter. A 5' deletion revealed a negative regulatory element located between bp -533 and -100 relative to the transcriptional start site. In the absence of this element, the promoter was still developmentally regulated but about fourfold more active. Also, the truncated promoter region retained normal dependence on two developmental regulators, FruA and DevS, but lost its dependence on the C-signaling protein CsgA. We infer that C signaling partially overcomes the negative effect of the upstream element on activity of the Omega4406 promoter. Deletion of downstream DNA between bp 50 and 140 caused a threefold loss in expression, suggesting that a positive regulatory element lies in this region. Additional positive and negative regulatory elements are present in the region from bp -69 to -49, based on the effects of multiple-base-pair mutations. Within this region, a 5-bp element and a C-box-like sequence resemble sequences found in other developmentally regulated M. xanthus promoter regions, but the effects of single-base-pair changes in these sequences suggest that each functions uniquely. We conclude that regulation of the Omega4406 promoter involves multiple positive and negative regulatory elements located upstream and downstream of the region typically bound by RNA polymerase.

The conserved kinase NHK-1 is essential for mitotic progression and unifying acentrosomal meiotic spindles in Drosophila melanogaster.

Conventional centrosomes are absent from the spindle in female meiosis in many species, but it is not clear how multiple chromosomes form one shared bipolar spindle without centrosomes. We identified a female sterile mutant in which each bivalent chromosome often forms a separate bipolar metaphase I spindle. Unlike wild type, prophase I chromosomes fail to form a single compact structure within the oocyte nucleus, although the integrity of metaphase I chromosomes appears to be normal. Molecular analysis indicates that the mutant is defective in the conserved kinase nucleosomal histone kinase-1 (NHK-1). Isolation of further alleles and RNA interference in S2 cells demonstrated that NHK-1 is also required for mitotic progression. NHK-1 itself is phosphorylated in mitosis and female meiosis, suggesting that this kinase is part of the regulatory system coordinating progression of mitosis and meiosis.

A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem.

The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as critical to haem-binding; the location of this histidine residue is similar to the cGMP-binding site in a crystal structure of cyclic nucleotide phosphodiesterase 2A. GAF domains are frequently involved in binding cyclic nucleotides, but this is the first GAF domain to be identified that binds haem. In contrast, PAS domains (similar to GAF domains in structure but not primary sequence) frequently use haem cofactors, and these findings further illustrate how the functions of these domains overlap. We propose that the activation of the DosS sensor is controlled through the haem binding of molecular oxygen or nitric oxide.

Nuclear receptor-dependent transcription with chromatin. Is it all about enzymes?

Nuclear receptors (NRs) are ligand-regulated, DNA-binding transcription factors that function in the chromatin environment of the nucleus to alter the expression of subsets of hormone-responsive genes. It is clear that chromatin, rather than being a passive player, has a profound effect on both transcriptional repression and activation mediated by NRs. NRs act in conjunction with at least three general classes of cofactors to regulate transcription in the context of chromatin: (a) chromatin remodelers; (b) corepressors; and (c) coactivators, many of which have distinct enzymatic activities that remodel nucleosomes or covalently modify histones (e.g. acetylases, deacetylases, methyltransferases, and kinases). In this paper, we will present a brief overview of these enzymes, their activities, and how they assist NRs in the repression or activation of transcription in the context of chromatin.

Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus.

During cell division complete DNA replication must occur before mitosis is initiated. Using a cell-free extract derived from Xenopus eggs that oscillates between S phase and mitosis, we have investigated how completion of DNA synthesis is coupled to the initiation of mitosis. We find that Xenopus eggs contain a feedback pathway which suppresses mitosis until replication is completed and that activation of this inhibitory system is dependent on the presence of a threshold concentration of unreplicated DNA. We demonstrate that in the presence of unreplicated DNA the active feedback system inhibits initiation of mitosis by blocking the activation of MPF, a regulator of mitosis found in all eukaryotic cells. Our results demonstrate that the feedback system does not inhibit MPF activation by blocking the synthesis or accumulation of cyclin protein, a subunit of MPF, or by blocking association of cyclin with the cdc2 subunit of MPF. We propose that the feedback system blocks mitosis by maintaining MPF in an inactive state by modulating posttranslational modifications critical for MPF activation.

Mammalian growth-associated H1 histone kinase: a homolog of cdc2+/CDC28 protein kinases controlling mitotic entry in yeast and frog cells.

Mammalian growth-associated H1 histone kinase, an enzyme whose activity is sharply elevated at mitosis, is similar to cdc2+ protein kinase from Schizosaccharomyces pombe and CDC28 protein kinase from Saccharomyces cerevisiae with respect to immunoreactivity, molecular size, and specificity for phosphorylation sites in H1 histone. Phosphorylation of specific growth-associated sites in H1 histone is catalyzed by yeast cdc2+/CDC28 kinase, as shown by the in vitro thermal lability of this activity in extracts prepared from temperature-sensitive mutants. In addition, highly purified Xenopus maturation-promoting factor catalyzes phosphorylation of the same sites in H1 as do the mammalian and yeast kinases. The data indicate that growth-associated H1 kinase is encoded by a mammalian homolog of cdc2+/CDC28 protein kinase, which controls entry into mitosis in yeast and frog cells. Since H1 histone is known to be an in vivo substrate of the mammalian kinase, this suggests that phosphorylation of H1 histone or an H1 histone counterpart is an important component of the mechanism for entry of cells into mitosis.

Protein kinases in neutrophils: a review.

In chemotactic factor-stimulated neutrophils, rapid increases of intracellular levels of cyclic AMP, calcium, and diacylglycerol have been observed and may be linked to protein kinase activation. The study of the physiological role and regulation of protein kinases in the neutrophil and the identification of their substrates has provided valuable information on the molecular mechanism of neutrophil activation. The focus of this review is on those aspects of protein kinases that are relevant to neutrophil activation and on the substrate proteins for these protein kinases. The possible role of protein phosphorylation in neutrophil function is also discussed.

Studies on the mechanism of action of the histone kinase dependent on adenosine 3',5'-monophosphate. Fast kinetics of histone H1 phosphorylation.

The transient phase of histone H1 phosphorylation was studied by the quenched-flow method. A 'minimal' kinetic scheme of the above process was proposed. A formal kinetic analysis was given to a four-step mechanism of the reaction. Computer simulation of the transient-phase kinetics of H1 phosphorylation and the steady-state kinetics of phosphate transfer from the enzyme phosphoform to histone permitted us to estimate all kinetic constants of the proposed mechanism.

Studies on the mechanism of action of the histone kinase dependent on adenosine 3',5'-monophosphate. Investigation of protein-protein interaction by electron spin-resonance spectroscopy and stopped-flow methods.

The interaction of the cAMP-dependent protein kinase catalytic subunit with its protein substrate, histone H1, was studied. The H1 molecule was specifically converted into the aminotyrosine-72 derivative. Fluorescent and spin labels were introduced into this residue. The changes in the ESR and fluorescence spectra of respective derivatives were observed upon the interaction of the latter with the catalytic subunit, thus enabling us to determine some kinetic and equilibrium parameters of this process. Stopped-flow investigation of the transient phase of the binding reaction indicates that the kinetic curve is described by a three-exponential function. The rate of protein-protein interaction is close to the rate of phosphate transfer from phosphoenzyme intermediate to protein substrate.