Mammalian target of rapamycin complex 2 (mTORC2) controls glycolytic gene expression by regulating Histone H3 Lysine 56 acetylation

Metabolic reprogramming is a hallmark of cancer cells, but the mechanisms are not well understood. The mammalian target of rapamycin complex 2 (mTORC2) controls cell growth and proliferation and plays a critical role in metabolic reprogramming in glioma. mTORC2 regulates cellular processes such as cell survival, metabolism, and proliferation by phosphorylation of AGC kinases. Components of mTORC2 are shown to localize to the nucleus, but whether mTORC2 modulates epigenetic modifications to regulate gene expression is not known. Here, we identified histone H3 lysine 56 acetylation (H3K56Ac) is regulated by mTORC2 and show that global H3K56Ac levels were downregulated on mTORC2 knockdown but not on mTORC1 knockdown. mTORC2 promotes H3K56Ac in a tuberous sclerosis complex 1/2 (TSC1/2) mediated signaling pathway. We show that knockdown of sirtuin6 (SIRT6) prevented H3K56 deacetylation in mTORC2 depleted cells. Using glioma model consisting of U87EGFRvIII cells, we established that mTORC2 promotes H3K56Ac in glioma. Finally, we show that mTORC2 regulates the expression of glycolytic genes by regulating H3K56Ac levels at the promoters of these genes in glioma cells and depletion of mTOR leads to increased recruitment of SIRT6 to these promoters. Collectively, these results identify mTORC2 signaling pathway positively promotes H3K56Ac through which it may mediate metabolic reprogramming in glioma.     

Kinetic mechanism of protein arginine methyltransferase 6 (PRMT6)

The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the mono- and dimethylation of arginine residues in a variety of proteins. Although these enzymes play important roles in a variety of cellular processes, aberrant PRMT activity is associated with several disease states, including heart disease and cancer. In an effort to guide the development of inhibitors targeting individual PRMTs, we initiated studies to characterize the molecular mechanisms of PRMT catalysis. Herein, we report studies on the kinetic mechanism of PRMT6. Initial velocity, product inhibition, and dead-end analog inhibition studies with the AcH4-21 and R1 peptides, as well as their monomethylated versions, indicate, in contrast to a previous report, that PRMT6 utilizes a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes.     

Toward convergence of experimental studies and theoretical modeling of the chromatin fiber

Understanding the structural organization of eukaryotic chromatin and its control of gene expression represents one of the most fundamental and open challenges in modern biology. Recent experimental advances have revealed important characteristics of chromatin in response to changes in external conditions and histone composition, such as the conformational complexity of linker DNA and histone tail domains upon compact folding of the fiber. In addition, modeling studies based on high-resolution nucleosome models have helped explain the conformational features of chromatin structural elements and their interactions in terms of chromatin fiber models. This minireview discusses recent progress and evidence supporting structural heterogeneity in chromatin fibers, reconciling apparently contradictory fiber models.     

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae

At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.     

The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body-P body pathway

Survival motor neuron protein (SMN) is the determining factor for the human neurodegenerative disease spinal muscular atrophy (SMA). SMN is critical for small nuclear ribonucleoprotein (snRNP) assembly. Using Drosophila oogenesis as a model system, we show that mutations in smn cause abnormal nuclear organization in nurse cells and oocytes. Germline and mitotic clonal analysis reveals that both nurse cells and oocytes require SMN to maintain normal organization of nuclear compartments including chromosomes, nucleoli, Cajal bodies and histone locus bodies. We previously found that SMN-containing U bodies invariably associate with P bodies (Liu, J. L., and Gall, J. G. (2007). U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies. Proc. Natl. Acad. Sci. U. S. A. 104, 11655-11659.). Multiple lines of evidence implicate SMN in the regulation of germline nuclear organization through the connection of U bodies and P bodies. Firstly, smn germline clones phenocopy mutations for two P body components, Cup and Ovarian tumour (Otu). Secondly, P body mutations disrupt SMN distribution and the organization of U bodies. Finally, mutations in smn disrupt the function and organization of U bodies and P bodies. Taken together, our results suggest that SMN is required for the functional integrity of the U body-P body pathway, which in turn is important for maintaining proper nuclear architecture.     

Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3′ cleavage reaction

The 3′ end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3′ untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3′ cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3′ cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved l-arginine β-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200 μM to the 200 nM–100 μM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3′ end formation.     

Identification of novel lysine demethylase 5-selective inhibitors by inhibitor-based fragment merging strategy

Histone lysine demethylases (KDMs) have drawn much attention as targets of therapeutic agents. KDM5 proteins, which are Fe(II)/α-ketoglutarate-dependent demethylases, are associated with oncogenesis and drug resistance in cancer cells, and KDM5-selective inhibitors are expected to be anticancer drugs. However, few cell-active KDM5 inhibitors have been reported and there is an obvious need to discover more. In this study, we pursued the identification of highly potent and cell-active KDM5-selective inhibitors. Based on the reported KDM5 inhibitors, we designed several compounds by strategically merging two fragments for competitive inhibition with α-ketoglutarate and for KDM5-selective inhibition. Among them, compounds 10 and 13, which have a 3-cyano pyrazolo[1,5-a]pyrimidin-7-one scaffold, exhibited strong KDM5-inhibitory activity and significant KDM5 selectivity. In cellular assays using human lung cancer cell line A549, 10 and 13 increased the levels of trimethylated lysine 4 on histone H3, which is a specific substrate of KDM5s, and induced growth inhibition of A549 cells. These results should provide a basis for the development of cell-active KDM5 inhibitors to highlight the validity of our inhibitor-based fragment merging strategy.     

Circular permuted proteins in the universe of protein folds

Finding and identifying circular permuted protein pairs (CPP) is one of the harder tasks for structure alignment programs, because of the different location of the break in the polypeptide chain connectivity. The protein structure alignment tool GANGSTA+ was used to search for CPPs in a database of nearly 10,000 protein structures. It also allows determination of the statistical significance of the occurrence of circular permutations in the protein universe. The number of detected CPPs was found to be higher than expected, raising questions about the evolutionary processes leading to CPPs. The GANGSTA+ protein structure alignment tool is available online via the web server at http://gangsta.chemie.fu‐berlin.de . On the same webpage the complete data base of similar protein structure pairs based on the ASTRAL40 set of protein domains is provided and one can select CPPs specifically. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Proteomic analysis identifies differentially expressed proteins after HDAC vorinostat and EGFR inhibitor gefitinib treatments in Hep-2 cancer cells

Several solid tumors are characterized by poor prognosis and few effective treatment options, other than palliative chemotherapy in the recurrent/metastatic setting. Epidermal growth factor receptor (EGFR) has been considered an important anticancer target because it is involved in the development and progression of several solid tumors; however, only a subset of patients show a clinically meaningful response to EGFR inhibition, particularly to EGFR tyrosine kinase inhibitors such as gefitinib. We have recently demonstrated synergistic antitumor effect of the histone deacetylase inhibitor vorinostat combined with gefitinib. To further characterize the interaction between these two agents, cellular extracts from Hep‐2 cancer cells that were untreated or treated for 24 h with either vorinostat or gefitinib alone or with a vorinostat/gefitinib combination were analyzed using 2‐D DIGE. Software analysis using DeCyder was performed, and numerous differentially expressed protein spots were visualized between the four examined settings. Using MALDI‐TOF MS and ESI‐Ion trap MS/MS, several differentially expressed proteins were identified; some of these were validated by Western blotting. Finally, a pathway analysis of experimental data performed using MetaCore highlighted a relevant relationship between the identified proteins and additional potential effectors. In conclusion, we performed a comprehensive analysis of proteins regulated by vorinostat and gefitinib, alone and in combination, providing a useful insight into their mechanisms of action as well as their synergistic interaction.