GCN5 dependence of chromatin remodeling and transcriptional activation by the GAL4 and VP16 activation domains in budding yeast.

Chromatin-modifying enzymes such as the histone acetyltransferase GCN5 can contribute to transcriptional activation at steps subsequent to the initial binding of transcriptional activators. However, few studies have directly examined dependence of chromatin remodeling in vivo on GCN5 or other acetyltransferases, and none have examined remodeling via nucleosomal activator binding sites. In this study, we have monitored chromatin perturbation via nucleosomal binding sites in the yeast episome TALS by GAL4 derivatives in GCN5(+) and gcn5Delta yeast cells. The strong activator GAL4 shows no dependence on GCN5 for remodeling TALS chromatin, whereas GAL4-estrogen receptor-VP16 shows substantial, albeit not complete, GCN5 dependence. Mini-GAL4 derivatives having weakened interactions with TATA-binding protein and TFIIB exhibit a strong dependence on GCN5 for both transcriptional activation and TALS remodeling not seen for native GAL4. These results indicate that GCN5 can contribute to chromatin remodeling at activator binding sites and that dependence on coactivator function for a given activator can vary according to the type and strength of contacts that it makes with other factors. We also found a weaker dependence for chromatin remodeling on SPT7 than on GCN5, indicating that GCN5 can function via pathways independent of the SAGA complex. Finally, we examine dependence on GCN5 and SWI-SNF at two model promoters and find that although these two chromatin-remodeling and/or modification activities may sometimes work together, in other instances they act in complementary fashion.

Artificially recruited TATA-binding protein fails to remodel chromatin and does not activate three promoters that require chromatin remodeling.

Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing the HIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at the CHA1 promoter. Finally, we show that activation of the GAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.

Mutations in the AF-2/hormone-binding domain of the chimeric activator GAL4.estrogen receptor.VP16 inhibit hormone-dependent transcriptional activation and chromatin remodeling in yeast.

GAL4.estrogen receptor.VP16 (GAL4.ER.VP16), which contains the GAL4 DNA-binding domain, the human ER hormone binding (AF-2) domain, and the VP16 activation domain, functions as a hormone-dependent transcriptional activator in yeast (Louvion, J.-F., Havaux-Copf, B., and Picard, D. (1993) Gene (Amst.) 131, 129-134). Previously, we showed that this activator can remodel chromatin in yeast in a hormone-dependent manner. In this work, we show that a weakened VP16 activation domain in GAL4.ER.VP16 still allows hormone-dependent chromatin remodeling, but mutations in the AF-2 domain that abolish activity in the native ER also eliminate the ability of GAL4.ER.VP16 to activate transcription and to remodel chromatin. These findings suggest that an important role of the AF-2 domain in the native ER is to mask the activation potential of the AF-1 activation domain in the unliganded state; upon ligand activation, a conformational change releases AF-2-mediated repression and transcriptional activation ensues. We also show that the AF-2 domain, although inactive at simple promoters on its own in yeast, can enhance transcription by the MCM1 activator in hormone-dependent manner, consistent with its having a role in activation as well as repression in the native ER.

Two domains of the beta subunit of neuronal nicotinic acetylcholine receptors contribute to the affinity of substance P.

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of alpha and beta subunits exhibited differential sensitivity to substance P, with those containing beta-4 subunits having a 25-fold higher affinity than those having beta-2 subunits. To identify the regions and/or amino acid residues of the beta subunit responsible for this difference, chimeric beta subunits were coexpressed with alpha-3 in Xenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (beta4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, beta4(V108)/beta2(F106), which is in the middle of the first extracellular domain, and beta4(F255)/beta2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the beta subunit-dependent differences in the receptor affinity for substance P.

Chromatin remodeling by transcriptional activation domains in a yeast episome.

We examine the generality of transcription factor-mediated chromatin remodeling by monitoring changes in chromatin structure in a yeast (Saccharomyces cerevisiae) episome outside of the context of a natural promoter. The episome has a well defined chromatin structure and a binding site for the transcription factor GAL4 but lacks a nearby functional TATA element or transcription start site, so that changes in chromatin structure are unlikely to be caused by transcription. To separate changes caused by binding and by activation domains, we use both GAL4 and a chimeric, hormone-dependent activator consisting of the GAL4 DNA-binding domain, an estrogen receptor (ER) hormone-binding domain, and a VP16 activation domain (Louvion, J.-F., Havaux-Copf, B. and Picard, D. (1993) Gene (Amst.) 131, 129-134). Both GAL4 and GAL4.ER.VP16 show very little perturbation of chromatin structure in their nonactivating configurations. Substantial additional perturbation occurs upon activation. This additional perturbation is marked by changes in micrococcal nuclease cleavage patterns, restriction endonuclease accessibility, and DNA topology and is not seen with the nonactivating derivative GAL4.ER. Remodeling by GAL4.ER.VP16 is detectable within 15 min following hormone addition and is complete within 45 min, suggesting that replication is not required. We conclude that activation domains can exert a major influence on chromatin remodeling by increasing binding affinity and/or by recruitment of other chromatin remodeling activities and that this remodeling can occur outside the context of a bona fide promoter.

The beta subunit of neuronal nicotinic acetylcholine receptors is a determinant of the affinity for substance P inhibition.

Substance P is known to inhibit nicotinic acetylcholine receptors from neuronal tissue, skeletal muscle, and electroplaque. The interaction of substance P with specific combinations of neuronal nicotinic acetylcholine receptor subunits was studied by expressing various combinations of subunits in Xenopus oocytes. The response to acetylcholine was inhibited by substance P with all subunit combinations tested; however, the apparent affinity for substance P varied by 20-30-fold. The affinity seemed to be dependent on the beta subtype expressed (beta 4 or beta 2). This suggests that the beta subunit may contribute, at least partially, to the substance P binding site. In the case of the alpha 7 subtype, which forms a homooligomeric receptor, the apparent affinity for substance P was intermediate between those of the two beta subtypes coexpressed with either alpha 3 or alpha 4. As previously found, the inhibition was noncompetitive. Furthermore, the inhibition was not voltage dependent and, therefore, is unlikely to be due to substance P blocking the channel within the transmembrane portion of the pore.

Binding of the dihydropyridine calcium channel blocker (+)-[3H] isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-3-pyridinecarboxylate (PN200-110) to RINm5F membranes and cells: characterization and functional significance.

This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200-110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200-110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+)-[3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites.