Although co-ordinate interaction between different signal transduction pathways is essential for developmental decisions, interpathway connections are often obscured and difficult to identify due to cross-talk. Here signals from the fission yeast stress-activated MAPK Spc1 are shown to regulate Cgs2, a negative regulator of the cAMP-dependent protein kinase (protein kinase A) pathway. Pathway integration is achieved via Spc1-dependent binding of Atf1-Pcr1 heterodimer to an M26 DNA site in the cgs2 ؉ promoter, which remodels chromatin to regulate expression of cgs2 ؉ and targets downstream of protein kinase A. This direct interpathway connection co-ordinates signals of nitrogen and carbon source depletion to affect a G 0 cell-cycle checkpoint and sexual differentiation. The Atf1-Pcr1-M26 complex-dependent chromatin remodeling provides a molecular mechanism whereby Atf1-Pcr1 heterodimer can function differentially as either a transcriptional activator, or as a transcriptional repressor, or as an inducer of meiotic recombination. We also show that the Atf1-Pcr1-M26 complex functions as both an inducer and repressor of chromatin remodeling, which provides a way for various chromatin remodeling-dependent effector functions to be regulated.

Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP (8-Br-cAMP) individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation (ChIP) showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; i.e., differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.

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DNA‐binding proteins present in fourth instar larvae of Anopheles stephensi were isolated by affinity chromatography on native and denatured DNA cellulose columns and analyzed by electrophoresis on polyacrylamide gels. A denatured DNA‐specific protein with an approximate molecular weight of 30 kDa was the predominant DNA binding protein of larvae. This protein was purified to electrophoretic homogeneity by ammonium sulfate fractionation followed by phosphocellulose chromatography. The purified 30 kDa binding protein showed an endonucleolytic activity capable of converting pBR 322 supercoiled DNA to the circular form. Maximum endonucleolytic activity was observed in the presence of 5 mM Mg2+ at pH 7.4. Enzyme activity was completely inhibited by EDTA.Arch. Insect Biochem. Physiol. 44:40–46, 2000. © 2000 Wiley‐Liss, Inc.

Although NR4A orphan nuclear receptors have been implicated in inflammatory gene expression and atherosclerosis, there is little information regarding their regulation by combinations of stimuli likely to be found in vivo, such as LPS and inducers of cAMP. LPS rapidly induced Nur77 and NOR1 mRNAs but had little effect on expression of Nurr1 mRNA. All three NR4As were rapidly induced by 8-bromo-cAMP and remained elevated for at least 8 h. Maximum induction of NOR1 by 8-bromo-cAMP was much greater than with LPS, but maximum induction of Nur77 was similar with both agents. Whereas Nurr1 mRNA had very little response to LPS, it was strongly induced by 8-bromo-cAMP, and Nurr1 mRNA levels remained elevated for at least 20 h. The combination of 8-bromo-cAMP and LPS acted synergistically to strongly induce NOR1 and Nur77, whereas LPS partially inhibited the induction of Nurr1 by 8-bromo-cAMP. Nurr1 protein levels correlated with Nurr1 mRNA levels but exhibited slower response kinetics. In summary, the NR4A receptors did not respond identically to individual stimuli or to combinations of stimuli. In particular, the magnitude of the responses to combinations of stimuli was quite different than to individual stimuli. Thus, NR4A receptor expression and the resulting functional consequences are more complex than appreciated from previous studies that evaluated regulation of NR4A expression by single stimuli.