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The non-steroidal anti-inflammatory drug Celecoxib is a specific inhibitor of cyclooxygenase-2. Apart from its inhibitor function, Celecoxib induces apoptosis through the intrinsic pathway which is controlled by the Bcl-2 family members. In Jurkat T lymphoma cells, treatment with Celecoxib results in a rapid decline of the anti-apoptotic Bcl-2-related protein Mcl-1. The depletion of Mcl-1 is sufficient for apoptosis induction and can be blocked by overexpression of Bcl-xL but not by the close homologue Bcl-2. The present investigation analyzed the mechanism by which Bcl-xL prevents apoptosis induction whereas Bcl-2 failed to. Our data show that the involvement of the orphan nuclear receptor Nur77/TR3 specifically targeting Bcl-2 but not Bcl-xL was not involved in Celecoxib-induced apoptosis. Surprisingly, BH3-only proteins Bid, Bim, and Puma of the Bcl-2 family were not needed either. However, unlike Bcl-2, Mcl-1, and Bcl-xL sequestered Bak preventing it from activation through a direct interaction. Thus, when abundantly expressed, Bcl-xL can substitute for the loss of Mcl-1 whereas Bcl-2, incapable of forming a high affinity complex with Bak, could not., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The histone variant H3.3 is implicated in the formation and maintenance of specialized chromatin structure in metazoan cells. H3.3-containing nucleosomes are assembled in a replication-independent manner by means of dedicated chaperone proteins. We previously identified the death domain associated protein (Daxx) and the alpha-thalassemia X-linked mental retardation protein (ATRX) as H3.3-associated proteins. Here, we report that the highly conserved N terminus of Daxx interacts directly with variant-specific residues in the H3.3 core. Recombinant Daxx assembles H3.3/H4 tetramers on DNA templates, and the ATRX-Daxx complex catalyzes the deposition and remodeling of H3.3-containing nucleosomes. We find that the ATRX-Daxx complex is bound to telomeric chromatin, and that both components of this complex are required for H3.3 deposition at telomeres in murine embryonic stem cells (ESCs). These data demonstrate that Daxx functions as an H3.3-specific chaperone and facilitates the deposition of H3.3 at heterochromatin loci in the context of the ATRX-Daxx complex.

Histone proteins often come in different variants serving specialized functions in addition to their fundamental role in packaging DNA. The metazoan histone H3.3 has been most closely associated with active transcription. Its role in histone replacement at active genes and promoters is conserved to the single histone H3 in yeast. However, recent genetic studies in flies have challenged its importance as a mark of active chromatin, and revealed unexpected insights into essential functions of H3.3 in the germline. With strikingly little amino acid sequence difference to the canonical H3, H3.3 therefore accomplishes a surprising variety of cellular and developmental processes., (2010 Elsevier Ltd. All rights reserved.)

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells., ((c) 2010 Elsevier Inc. All rights reserved.)

Histone H3.3 is a universal replacement histone in metazoans that has been implicated in diverse processes ranging from gene activation to heterochromatin silencing. Here, we show that, before deposition, H3.3 exists in two biochemically distinct complexes, associated with either Daxx or HIRA, Ubinuclein-1, and Cabin-1. Although the HIRA complex is evolutionarily conserved in yeast, Daxx is a novel histone chaperone unique to metazoans. Deletion of HIRA in mouse embryonic stem cells impairs the HIRA complex integrity but does not abolish Daxx association with H3.3/H4. Similarly, HIRA interacts with H3.3/H4 in the absence of Daxx. We hypothesize that these two H3.3 chaperone systems provide separate pools of H3/H4 units for incorporation at distinct sites within the genome. We provide evidence that the association of histone H3.3 with distinct assembly systems allows it to acquire unique posttranslational modifications before deposition that might affect its role after incorporation into chromatin.

The cyclooxygenase-2 inhibitor Celecoxib is a potent inducer of apoptosis in tumor cells. In most cellular systems Celecoxib induces apoptosis via an intrinsic, mitochondrial apoptosis pathway. We recently showed that in Bax-negative Jurkat cells expression of pro-apoptotic Bak is essential for Celecoxib-induced mitochondrial damage and apoptosis induction. Aim of the present study was to identify specific pro- and anti-apoptotic members of the Bcl-2 family involved in the regulation of Bak activation, and subsequent apoptosis upon treatment with Celecoxib in the Jurkat cell model. Our results show that apoptosis in response to Celecoxib required the presence of Noxa and downregulation of the anti-apoptotic protein Mcl-1. Celecoxib-induced Bak activation and subsequent apoptosis could be inhibited by overexpression of Bcl-xL but not by the very similar Bcl-2. In Bcl-xL-overexpressing cells neutralization of both, Mcl-1 and Bcl-xL, was prerequisite for an efficient induction of apoptosis. Our data reveal an important role of the Mcl-1/Noxa axis for Celecoxib-induced apoptosis and suggest that Celecoxib may be of value for treatment of tumors addicted to Mcl-1 and for combined treatment approaches targeting anti-apoptotic Bcl-2 family members.

The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib constitutes the prototype of pro-apoptotic agents acting through the intrinsic death pathway in a Bcl-2 independent manner. To gain further insight into celecoxib-mediated apoptosis regulation at the level of the mitochondria we tested in how far the crucial pro-apoptotic Bcl-2 proteins Bak and Bax were involved using clones of the Bax-deficient Jurkat T-lymphoma cell model either expressing Bak (Jurkat Bak positive) or being negative for Bak (Jurkat Bak negative), or overexpressing Bcl-2 (Jurkat Bcl-2). Celecoxib induced substantial apoptosis in Jurkat Bak-positive cells. Overexpression of Bcl-2 had only limited protective effects. However, loss of Bak-expression conferred almost complete resistance of Jurkat cells to celecoxib-induced apoptosis. Neither enhanced celecoxib-concentrations nor prolonged incubation times were sufficient to normalize apoptotic rates upon celecoxib-treatment in these Bak/Bax-negative cells. In line with that observation, siRNA-mediated silencing of Bak in the Bak-positive Jurkat cells largely reduced the extent of celecoxib-induced cell death. Interestingly, in celecoxib-sensitive Bak-positive cells, celecoxib-treatment resulted in down-regulation of the anti-apoptotic Bcl-2 protein Mcl-1 which may contribute to celecoxib-mediated activation of Bak-dependent apoptosis. Taken together our data clearly show for the first time the functional relevance of Bak for celecoxib-induced apoptosis in Bax-deficient Jurkat T-lymphoma cells.

Although the biological significance of protein phosphorylation in cellular signaling is widely appreciated, methods to directly detect these post-translational modifications in situ are lacking. Here we introduce the application of high-resolution NMR spectroscopy for observing de novo protein phosphorylation in vitro and in Xenopus laevis egg extracts and whole live oocyte cells. We found that the stepwise modification of adjacent casein kinase 2 (CK2) substrate sites within the viral SV40 large T antigen regulatory region proceeded in a defined order and through intermediate substrate release. This kinase mechanism contrasts with a more intuitive mode of CK2 action in which the kinase would remain substrate bound to perform both modification reactions without intermediate substrate release. For cellular signaling pathways, the transient availability of partially modified CK2 substrates could exert important switch-like regulatory functions.