Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

Abstract

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.Binding of transcriptional activators to DNA is frequently inhibited by the presence of nucleosomes. GAL4 derivatives, glucocorticoid receptor, progesterone receptor, USF, Spl, and Myc/Max heterodimers are all able to bind to nucleosomal DNA, but the affinity of these activators for nucleosomal DNA is decreased by up to three orders of magnitude over that observed on naked DNA (1-12). Other activators, such as NF1 and human heat shock factor 1, cannot bind to nucleosomal DNA under conditions tested so far, raising the possibility that nucleosomal inhibition of binding might prevent the function of these activators under certain circumstances (3,10,13). The mechanisms that facilitate activator binding to chromatin are therefore a key aspect of transcriptional control in eukaryotes.Several considerations suggest that activation domains might increase the ability of activators to bind to nucleosomal DNA. Activation domains can contact general transcription factors (GTFs; for review, see refs. 14 and 15), leading to the possibility that cooperative interactions between activators and GTFs might facilitate activator binding in an activationdomain-dependent manner. Activation domains are also capable of alleviating the repressive effects of nucleosomes on transcription in vitro (16-18), and can alter chromatin structure in intact yeast cells (19,20 We demonstrate here that the VP16 activation domain can enhance the ability of the corresponding GAL4 derivative to bind to sites located in genomic chromatin in mammalian cells. In addition, we have measured the relative activation potentials of these GAL4 derivatives and others containing different activation domains on chromosomal and transiently transfected reporters. We found that the relative potencies of these activators were dramatically different when compared under these two conditions, such that only certain activation domains were effective in stimulating t...

GCN5 Dependence of Chromatin Remodeling and Transcriptional Activation by the GAL4 and VP16 Activation Domains in Budding Yeast

“…For example, GAL4 perturbs a positioned nucleosome containing its binding site in yeast to a much greater degree in cells grown in galactose medium, when it is in its activating configuration, than in glucose, when it is not, and the chimeric activator GAL4-estrogen receptor (ER)-VP16 similarly is much more effective at this perturbation in the presence of activating ␤-estradiol than in its absence (66). Consistent with these results, binding of transcription factors is increased by ADs in both yeast and mammalian cells to sites having uncharacterized chromatin structure (9,70).…”

Enhancer Sequences Influence the Role of the Amino-Terminal Domain of Bicoid in Transcription

“…In particular, Bcd(A52-56), which has a stronger activation potential, can exert a dominant effect in wild-type embryos, causing a posterior shift of the target genes (38). These results suggest that a stronger Bcd can activate transcription at a lower concentration (or with fewer DNAbound molecules) in embryos, a concept that has been proposed previously for various activators (2,5,18,20,29). We propose that the muted self-inhibitory function of Bcd on the kni enhancer contributes to the protein's ability to activate transcription at low concentrations.…”

SWI-SNF Complex Participation in Transcriptional Activation at a Step Subsequent to Activator Binding

“…One way this could be reconciled with the present work is if stable binding to weak sites were especially dependent on the activation domain. Indeed, evidence exists that transcription factor binding and chromatin perturbation depends on activation domains in vivo (6,(69)(70)(71). Furthermore, stable binding of GAL4 to low-and moderate-affinity binding sites has been shown to be strengthened by the presence of a nearby TATA element (76).…”

“…This large complex, of about 2 ϫ 10 6 Da, contains at least 11 distinct polypeptides (8,61,72) and is conserved across most of the eukaryotic kingdom (60,80). Transcriptional induction of a variety of genes is impaired in yeast cells lacking components of the SWI-SNF complex (62,87), and genetic and biochemical evidence suggests that the SWI-SNF complex may aid transcriptional induction by helping to counteract the repressive effects of packaging DNA into chromatin.…”