The Kinases MSK1 and MSK2 Are Required for Epidermal Growth Factor-induced, but Not Tumor Necrosis Factor-induced, Histone H3 Ser 10 Phosphorylation

Phosphorylation of histone H3 protein at serine 10 is an important step in chromatin remodeling during transcriptional transactivation. I[kappa]B kinase-[alpha] (IKK-[alpha]) and Mitogen- and Stress-activated protein Kinases 1 and 2 (MSK1/2) have been shown to play key roles in the transcriptional regulation of immediate early genes such as c-fos. Interestingly, IKK-[alpha] and MSK1/2 have also been implicated as histone H3-Ser¹⁰ kinases. In this work, we have shown that MSK1/2 are required for epidermal growth factor (EGF)-induced, but not tumor necrosis factor-induced, histone H3-Ser¹⁰ phosphorylation, both globally and at specific promoters. Consistent with this, MSK1/2 are required for optimal immediate early c-fos transcription in response to EGF potentially through control of both H3-Ser¹⁰ and promoter-associated cAMP-response element-binding protein phosphorylation. Furthermore, MSK1/2 control EGF-induced I[kappa]B[alpha] promoter H3-Ser¹⁰ phosphorylation in the absence of elevated transcription. These studies demonstrate the existence of pathway-specific mechanisms to control histone H3-Ser¹⁰ phosphorylation and gene expression

A nucleosomal function for IκB kinase-α in NF-κB-dependent gene expression

NF-κB is a principal transcriptional regulator of diverse cytokine-mediated processes and is tightly controlled by the IκB kinase complex (IKK-α/β/γ). IKK-β and IKK-γ are critical for cytokine-induced NF-κB function, whereas IKK-α is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-α in NF-κB-dependent gene expression in response to cytokine treatment. Here we demonstrate nuclear accumulation of IKK-α after cytokine exposure, suggesting a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-α was recruited to the promoter regions of NF-κB-regulated genes on stimulation with tumour-necrosis factor-α. Notably, NF-κB-regulated gene expression is suppressed by the loss of IKK-α and this correlates with a complete loss of gene-specific phosphorylation of histone H3 on serine 10, a modification previously associated with positive gene expression. Furthermore, we show that IKK-α can directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. We propose that IKK-α is an essential regulator of NF-κB-dependent gene expression through control of promoter-associated histone phosphorylation after cytokine exposure. These findings provide additional insight into the role of the IKK complex in NF-κB-regulated gene expression

The Putative Oncoprotein Bcl-3 Induces Cyclin D1 To Stimulate G1 Transition

Bcl-3 is a distinctive member of the IκB family of NF-κB inhibitors because it can function to coactivate transcription. A potential involvement of Bcl-3 in oncogenesis is highlighted by the fact that it was cloned due to its location at a breakpoint junction in some cases of human B-cell chronic lymphocytic leukemia and that it is highly expressed in human breast tumor tissue. To analyze the effects of Bcl-3 dysregulation in breast epithelial cells, we created stable immortalized human breast epithelial cell lines either expressing Bcl-3 or carrying the corresponding vector control plasmid. Analysis of the Bcl-3-expressing cells suggests that these cells have a shortened G1 phase of the cell cycle as well as a significant increase in hyperphosphorylation of the retinoblastoma protein. Additionally, the cyclin D1 gene was found to be highly expressed in these cells. Upon further analysis, Bcl-3, acting as a coactivator with NF-κB p52 homodimers, was demonstrated to directly activate the cyclin D1 promoter through an NF-κB binding site. Therefore, our results demonstrate that dysregulated expression of Bcl-3 potentiates the G1 transition of the cell cycle by stimulating the transcription of the cyclin D1 gene in human breast epithelial cells

Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability

Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-κB-dependent Genes

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD

Recruitment and Activation of RSK2 by HIV-1 Tat

The transcriptional activity of the integrated HIV provirus is dependent on the chromatin organization of the viral promoter and the transactivator Tat. Tat recruits the cellular pTEFb complex and interacts with several chromatin-modifying enzymes, including the histone acetyltransferases p300 and PCAF. Here, we examined the interaction of Tat with activation-dependent histone kinases, including the p90 ribosomal S6 kinase 2 (RSK2). Dominant-negative RSK2 and treatment with a small-molecule inhibitor of RSK2 kinase activity inhibited the transcriptional activity of Tat, indicating that RSK2 is important for Tat function. Reconstitution of RSK2 in cells from subjects with a genetic defect in RSK2 expression (Coffin-Lowry syndrome) enhanced Tat transactivation. Tat interacted with RSK2 and activated RSK2 kinase activity in cells. Both properties were lost in a mutant Tat protein (F38A) that is deficient in HIV transactivation. Our data identify a novel reciprocal regulation of Tat and RSK2 function, which might serve to induce early changes in the chromatin organization of the HIV LTR

Increased IKKα Expression in the Basal Layer of the Epidermis of Transgenic Mice Enhances the Malignant Potential of Skin Tumors

Non-melanoma skin cancer is the most frequent type of cancer in humans. In this study we demonstrate that elevated IKKα expression in murine epidermis increases the malignancy potential of skin tumors. We describe the generation of transgenic mice overexpressing IKKα in the basal, proliferative layer of the epidermis and in the outer root sheath of hair follicles. The epidermis of K5-IKKα transgenic animals shows several alterations such as hyperproliferation, mislocalized expression of integrin-α6 and downregulation of the tumor suppressor maspin. Treatment of the back skin of mice with the mitogenic agent 12-O-tetradecanoylphorbol-13-acetate causes in transgenic mice the appearance of different preneoplastic changes such as epidermal atypia with loss of cell polarity and altered epidermal tissue architecture, while in wild type littermates this treatment only leads to the development of benign epidermal hyperplasia. Moreover, in skin carcinogenesis assays, transgenic mice carrying active Ha-ras (K5-IKKα-Tg.AC mice) develop invasive tumors, instead of the benign papillomas arising in wild type-Tg-AC mice also bearing an active Ha-ras. Therefore we provide evidence for a tumor promoter role of IKKα in skin cancer, similarly to what occurs in other neoplasias, including hepatocarcinomas and breast, prostate and colorectal cancer. The altered expression of cyclin D1, maspin and integrin-α6 in skin of transgenic mice provides, at least in part, the molecular bases for the increased malignant potential found in the K5-IKKα skin tumors

A nuclear phylogenomic study of the angiosperm order Myrtales, exploring the potential and limitations of the universal Angiosperms353 probe set

Premise: To further advance the understanding of the species-rich, economically and ecologically important angiosperm order Myrtales in the rosid clade, comprising nine families, approximately 400 genera and almost 14,000 species occurring on all continents (except Antarctica), we tested the Angiosperms353 probe kit. Methods: We combined high-throughput sequencing and target enrichment with the Angiosperms353 probe kit to evaluate a sample of 485 species across 305 genera (76 of all genera in the order). Results: Results provide the most comprehensive phylogenetic hypothesis for the order to date. Relationships at all ranks, such as the relationship of the early-diverging families, often reflect previous studies, but gene conflict is evident, and relationships previously found to be uncertain often remain so. Technical considerations for processing HTS data are also discussed. Conclusions: High-throughput sequencing and the Angiosperms353 probe kit are powerful tools for phylogenomic analysis, but better understanding of the genetic data available is required to identify genes and gene trees that account for likely incomplete lineage sorting and/or hybridization events

A nuclear phylogenomic study of the angiosperm order Myrtales, exploring the potential and limitations of the universal Angiosperms353 probe set

PREMISE: To further advance the understanding of the species- rich, economically and ecologically important angiosperm order Myrtales in the rosid clade, comprising nine families, approximately 400 genera and almost 14,000 species occurring on all continents (except Antarctica), we tested the Angiosperms353 probe kit.METHODS: We combined high- throughput sequencing and target enrichment with the Angiosperms353 probe kit to evaluate a sample of 485 species across 305 genera (76% of all genera in the order).RESULTS: Results provide the most comprehensive phylogenetic hypothesis for the order to date. Relationships at all ranks, such as the relationship of the early-diverging families, often reflect previous studies, but gene conflict is evident, and relationships previously found to be uncertain often remain so. Technical considerations for processing HTS data are also discussed.CONCLUSIONS: High- throughput sequencing and the Angiosperms353 probe kit are powerful tools for phylogenomic analysis, but better understanding of the genetic data available is required to identify genes and gene trees that account for likely incomplete lineage sorting and/or hybridization events