Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

Abstract

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.Transcription of eukaryotic protein-coding genes is controlled by the combinatorial arrangement of regulatory proteins that bind specific DNA elements located either upstream or downstream of a core promoter. Structural and functional analyses have revealed that sequence-specific activators are modular in structure. They most often contain at least two generally independent functional regions: a DNA-binding domain and a transcriptional activation domain, in addition to oligomerization and nuclear localization determinants. Four main distinct types of activation motifs, tentatively grouped according to their different amino acid compositions, have been identified thus far: the acidic negatively charged domains, the glutamine-rich domains, the proline-rich domains (34, 37), and the metal-binding cysteine-containing activating domain of the adenovirus Ela protein (29).Activation domains are thought to function by interacting directly or indirectly with general components of the transcription initiation machinery (26,38). This hypothesis is supported by the observation that an activator present at an artificially high intracellular concentration can inhibit its own transcriptional activity as well as that of other activators; this inhibition was previously called squelching or transcriptional interference (12,29,33,46,48). Squelching is thought to occur by the titration of a limiting soluble cellular component of the transcriptional machinery (the target) through its interaction with the activation domain of the overexpressed activator. Thus, squelching may result in the sequestration of the target, which can no longer interact with promoter-bound activators to mediate their transcriptional activation functions (37,38 synergistically with other activators recog...

The 30‐kDa rat liver transcription factor nuclear factor 1 binds the rat growth‐hormone proximal silencer

“…Members of this family were reported to act synergistically or to interact cooperatively with other transcriptional modulators, such as the estrogen receptor [36], the hepatocyte nuclear factor 3a [37], or an uncharacterized CAMP-responsive element-binding protein termed CRF [38]. Evidence was also found for NF1 being involved in mediating the transcriptional activation of the mouse a2(I) collagen promoter by transforming growth factor-j3 [ 171.…”

Estrogen Receptor Reduces CYP1A1 Induction in Cultured Human Endometrial Cells

“…Transcription of CYP1A1 is regulated, in part, by the NF-1 transcription factor, but CYP1B1 is not (56). In addition, NF-1 functions synergistically with estrogen receptor to activate transcription driven by that receptor (59). Because the estrogen receptor and NF-1 interact, we hypothesized that activated estrogen receptor may sequester NF-1 thereby reducing ability of the Ah receptor complex to induce CYP1A1.…”

Regulation of the Transforming Growth Factor β-responsive Transcription Factor CTF-1 by Calcineurin and Calcium/ Calmodulin-dependent Protein Kinase IV

“…Gel Mobility Shift Assays-For gel shift analysis of the endogenous CTF/NF-I or of the transiently expressed GAL4 fusion proteins, cells were lysed in extraction buffer (20 mM Tris, pH 7.5, 20% glycerol, 500 mM KCl, 1 mM dithiothreitol, and protease inhibitors) as described by Martinez et al (26). Whole-cell lysates were normalized for protein concentration and incubated with end-labeled double-stranded DNA probes containing either the high-affinity CTF/NF-I binding site found within the first 50 base pairs of the Adenovirus origin of replication (27) or the 17-base pair GAL4 binding site (28).…”