No abstract

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.

Promoter-and enhancer-binding factors appear to function by facilitating the transcription reaction as well as by counteracting chromatin-mediated repression (antirepression). We have examined the mechanism by which a hybrid activator, GAL4-VP16, is able to counteract histone HI-mediated repression by using both H1-DNA complexes and reconstituted HI-containing chromatin templates. The GAL4 DNA bind{ng domain alone was sufficient to disrupt local H1-DNA interactions, but a transcriptional activation region was additionally necessary for antirepression. GAL4--VP16-mediated antirepression required an auxiliary factor, denoted as a co-antirepressor, which was partially purified from Drosophila embryos. We have found that the co-antirepressor activity was sensitive to digestion with RNase A. Moreover, total RNA from Drosophila embryos could partially substitute for the co-antirepressor fraction, which indicated that the co-antirepressor may function as a histone acceptor ("histone sink"}. These findings suggest a model for gene activation in which sequence-specific transcription factors disrupt H1-DNA interactions at the promoter to facilitate transfer of HI to a histone acceptor, which then allows access of the basal transcription factors to the DNA template.[Key Words: Transcriptional regulation; histone H1; chromatin; RNA polymerase II; in vitro transcription] Received July 16, 1992; revised version accepted September 29, 1992.The proper control of gene expression is essential for the development, growth, and sustenance of eukaryotic organisms, yet the strategies and mechanisms by which genes are regulated remain to be clarified. An early step in the pathway leading to gene expression is initiation of transcription. Synthesis of mRNA is carried out by the RNA polymerase II transcriptional machinery, which comprises RNA polymerase II and several auxiliary factors that are commonly referred to as general factors (for recent reviews, see Saltzman and Weinmann 1989;Sawadogo and Sentenac 1990;Conaway and Conaway 1991; Zawel and Reinberg 1992). Transcription by the basal transcriptional apparatus is regulated by sequencespecific DNA-binding factors that interact with promoter and enhancer elements (for review, see Johnson and McKnight 1989;Mitchell and Tjian 1989), and it presently appears that many of these promoter-and enhancer-binding proteins may stimulate transcription by acting in conjunction with another class of factors that are referred to as coactivators, mediators, adaptors, or intermediary factors {for review, see Lewin 1990; Ptashne and Gann 1990; Pugh and Tjian 1992}. Transcriptional activity is also affected by chromatin structure {for review, see Weintraub 1985;Elgin 1988;Gross and Garrard 1988; van Holde 1989;Grunstein 1990;Wolffe 1990Wolffe , 1992 Felsenfeld 1992}; thus, it is important to consider the function of the general transcriptional machinery, the promoter-and enhancer-binding factors, and the coactivators with the chromatin template. 1991;To study the relationship between chromatin structure ...

We have examined the effect of HMG17 on transcription by RNA polymerase II by the assembly and analysis of HMG17-containing chromatin templates consisting of regularly spaced nucleosomal arrays. Structural analysis of the chromatin indicated that HMG17 is incorporated into chromatin in a physiological manner with the full complement of core histones. The transcriptional studies revealed that HMG17 stimulates transcription in conjunction with the sequence-specific activator GAL4-VP16. This effect was observed with chromatin, but not with non-nucleosomal templates, and required the presence of HMG17 during chromatin assembly. The incorporation of HMG17 into chromatin resulted in a 7-to 40-fold stimulation of GAL4-VP16-activated transcription to levels that were comparable to those observed with histone-free DNA templates. In contrast, transcription from HMG17-containing chromatin was not detectable in the absence of GAL4--VP16 or with a GAL4 derivative [GAL4(1-147)] lacking the VP16 activation domain. Finally, the incorporation of HMG17 into chromatin was found to increase the efficiency of transcription initiation, but not the extent of transcriptional elongation. Thus, HMG17 is a chromatin-specific transcriptional coactivator that increases the efficiency of initiation of transcription by RNA polymerase II.