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As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

Structural data of integrin alpha IIb beta 3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a "bent," low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that "clasping" the alpha IIb head domain to the beta 3 tail decreased fibrinogen binding. To study the role of alpha IIb extension about the genu, we introduced a disulfide "clamp" between the alpha IIb thigh and calf-1 domains. Clamped alpha IIb beta 3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn(2+) or by coexpressing the clamped alpha IIb with a beta 3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M(r) 7,500) or alpha IIb beta 3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAbAP5 in the presence of kistrin. Collectively, our studies support a role for alpha IIb extension about the genu in the binding of ligands of 340,000 and 900,000 M(r) with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with alpha IIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in beta 3 that affect the intrinsic affinity of the binding pocket for ligand.

Squamous cell carcinoma (SCC) is the most frequent cancer in organ transplant recipients (OTRs). The immune system plays a major role in the fight against SCC, however, little is known about the local inflammatory response in SCC at all. We analyzed quantity and quality of the perineoplastic inflammatory SCC microenvironment in immunocompetent patients and immmuno-suppressed OTRs. RNA expression profile of SCC patients was analyzed for 8 different sets of genes relating to Th1 versus Th2 response using Gene Set Enrichment Analysis. SCC from immunocompetent patients and OTRs were analyzed by real-time polymerase chain reactions for CD4, CD8, TBET, GATA-3, FOXP3, RORC, IFN-g, IL-4, TGF-beta, IL-10, and IL-17A mRNA expression. Immunohistochemistry was carried out in SCC for CD3, CD4, CD8, and FOXP3 expression. Considerable inflammation was seen in both patient groups. SCC in immunocompetent patients and OTRs was associated with a mixed Th1 and Th2 gene expression signature. CD4(+) mRNA was diminished in immunosuppression. Skin adjacent to SCC in OTRs showed Th2 expression pattern as compared with immunocompetent patients. T-BET and IFN-g mRNA expression were decreased in the OTR group. Although Th17-weighted inflammation was unchanged, IL-17A mRNA level was markedly decreased with immunosuppression. Regulatory T cells, characterized by FOX-P3 and TGF-beta mRNA level, were decreased in OTRs. Our findings support the hypothesis that nontumor-bearing skin adjacent to SCC in OTRs is not necessarily normal and that the local microenvironment may contribute to a field effect contributing to higher recurrence rates and more aggressive behavior observed in these patients.

MLL1 fusion proteins activate HoxA9 gene expression and cause aggressive leukemias that respond poorly to treatment, but how they recognize and stably bind to HoxA9 is not clearly understood. In a systematic analysis of MLL1 domain recruitment activity, we identified an essential MLL1 recruitment domain that includes the CXXC domain and PHD fingers and is controlled by direct interactions with the PAF elongation complex and H3K4Me2/3. MLL1 fusion proteins lack the PHD fingers and require prebinding of a wild-type MLL1 complex and CXXC domain recognition of DNA for stable HoxA9 association. Together, these results suggest that specific recruitment of MLL1 requires multiple interactions and is a precondition for stable recruitment of MLL1 fusion proteins to HoxA9 in leukemogenesis. Since wild-type MLL1 and oncogenic MLL1 fusion proteins have overlapping yet distinct recruitment mechanisms, this creates a window of opportunity that could be exploited for the development of targeted therapies.

Neuronal migration leads to a highly organized laminar structure in the mammalian brain, and its misregulation causes lissencephaly and behavioral and cognitive defects. Reelin signaling, which is mediated in part by a key adaptor, disabled-1 (Dab1), plays a critical but incompletely understood role in this process. We found that the neuron-specific RNA-binding protein Nova2 regulates neuronal migration in late-generated cortical and Purkinje neurons. An unbiased HITS-CLIP and exon junction array search for Nova-dependent reelin-pathway RNAs at E14.5 revealed only one candidate-an alternatively spliced isoform of Dab1 (Dab1.7bc). In utero electroporation demonstrated that Dab1.7bc was sufficient to induce neuronal migration defects in wild-type mice and exacerbate defects when Dab1 levels were reduced, whereas Dab1 overexpression mitigates defects in Nova2 null mice. Thus, Nova2 regulates an RNA switch controlling the ability of Dab1 to mediate neuronal responsiveness to reelin signaling and neuronal migration, suggesting new links between splicing regulation, brain disease, and development.

We present the results of a search for pair production of the supersymmetric partner of the top quark (the top squark (t) over tilde (1)) decaying to a b quark and a chargino (chi) over tilde (+/-)(1) with a subsequent (chi) over tilde (+/-)(1) decay into a neutralino (chi) over tilde (0)(1), lepton l, and neutrino nu Using a data sample corresponding to 2.7 fb(-1) of integrated luminosity of p (p) over bar collisions at root s = 1: 96 TeV collected by the CDF II detector, we reconstruct the mass of top squark candidate events and fit the observed mass spectrum to a combination of standard model processes and (t) over tilde (1)(t) over tilde (1). We find no evidence for (t) over tilde (1)(t) over tilde (1) production and set 95% C. L. limits on the masses of the top squark and the neutralino for several values of the chargino mass and the branching ratio B((X) over tilde (+/-)(1) -> (chi) over tilde (0)(1)l(+/-)nu).

Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants(1-6). In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive(1,7,8). Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes(1-4). Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1-4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail adynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.

This article starts in Part I with a simple example of two biochemical reaction networks that are indistinguishable at the macroscopic level but are different at the molecular level and are shown to have significantly different kinetic properties. So, if one completely ignores the fact that reactions advance in discrete steps at the molecular level, then one can fail to distinguish between networks with widely different kinetics. In part II biochemical reaction networks are treated in a general way to discover what property of a network, only seen at the molecular level, affects its kinetics. It is shown that every such network has a unique torsion group which can be described numerically and readily determined by a programmable computation. If the group is found to be the singleton {0} (as is most often the case in practice), then the network is said to be torsion-free and its kinetic properties unaffected by ignoring its discrete character. A chemical reaction network has to be represented algebraically to calculate its torsion group. If the network is to be understood only at the macroscopic level, it can be placed in the context of real vector spaces, but to recognize its discrete character and its torsion group, each vector space is replaced by a discrete subset of that space, where each molecule can be recognized as a distinct and indivisible entity. Next, the process of calculating a torsion group is shown in several cases, including the example in part I. In this particular case it is shown to have the torsion group with 2 elements, reflecting the fact that the substrate molecules become product molecules 2 at a time, with the result that the overall macroscopic reaction is R a double dagger" T, whereas at the molecular level it is 2R a double dagger" 2T. In general, however, the torsion group of a biochemical reaction network can be any finite additive group, which is a property of the network that can only be seen at the molecular level. Finally, this fact is demonstrated by showing how to construct a hypothetical, but plausible, biochemical reaction network that has any given finite additive group as its torsion group.

Steroidogenic acute regulatory protein (StAR)D4 is a member of the StAR related lipid transfer family. Homology comes from the similar to 210 amino acid lipid binding domain implicated in intracellular transport, cell signaling, and lipid metabolism. StARD4 was identified as a gene downregulated 2-fold by dietary cholesterol (Soccio, R. E., R. M. Adams, K. N. Maxwell, and J. L. Breslow. 2005. Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. J. Biol. Chem. 280: 19410-19418). A mouse knockout was created to investigate StARD4's functionality and role in lipid metabolism. Homozygous knockout mice exhibited normal Mendelian mating genetics, but weighed less than wild- type littermates, an effect not accounted for by energy metabolism or food intake. Body composition as analyzed by DEXA scan showed no significant difference. No significant alterations in plasma or liver lipid content were observed on a chow diet, but female knockout mice showed a decrease in gallbladder bile cholesterol and phospholipid concentration. When challenged with a 0.2% lova-statin diet, StARD4 homozygous mice exhibited no changes. However, when challenged with a 0.5% cholesterol diet, female StARD4 homozygous mice showed a moderate decrease in total cholesterol, LDL, and cholesterol ester concentrations. Microarray analysis of liver RNA found few changes. However, NPC1's expression, a gene not on the microarray, was decreased similar to 2.5-fold in knockouts. These observations suggest that StARD4's role can largely be compensated for by other intracellular cholesterol transporters.-Riegelhaupt, J. J., M. P. Waase, J. Garbarino, D. E. Cruz, and J. L. Breslow. Targeted disruption of steroidogenic acute regulatory protein D4 leads to modest weight reduction and minor alterations in lipid metabolism. J. Lipid Res. 2010. 51: 1134-1143.