Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter.

Abstract

We characterized the activity of a human hsp7O gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp7O promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp7O gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.Promoters recognized by RNA polymerase II require cis-acting regulatory elements both for constitutive activity and for modulation of transcription by various regulatory processes characteristic of different genes. These elements include sequences near the start of transcription, such as the TATA box, various upstream sequences in the region from approximately -50 to -200, and enhancer sequences that may be located at much greater distances either 5' or 3' to the start site. The mechanisms by which these elements act to control gene expression at the level of transcription are of great interest. One valuable approach to this problem is the identification and purification of sequence-specific DNAbinding proteins that interact with promoter and enhancer elements (9, 25).One regulatory system that has attracted attention is heat shock induction (23). The response of organisms to abnormally high temperature or to a variety of other metabolic stresses involves a rapid and dramatic increase in the rate of transcription initiation of several specific genes encoding heat shock proteins. Control also occurs at the level of mRNA splicing and translation in some, but not all, instances. This heat shock or stress response is very general, and perhaps universal, among eucaryotic organisms. Some of the heat shock gene products, such as the 70-kilodalton family, show remarkable amino acid sequence conservation (23). Perhaps more surprisingly, a 14-base-pair (bp) DNA sequence element, originally identified upstream of several Drosophila heat shock genes, also occurs with a high degree of homology in organisms as distantly related as yeasts and humans (23,28). In addition, the Drosophila hsp70 gene promoter is functional and responsive to heat shock induction in mammalian cells (28,29).

Genomic Mechanisms of p210BCR-ABL Signaling

“…Hsp70 induction is largely regulated at the transcriptional level in response to heat shock and mitogenic agents (41,42). The major identified regulatory site controlling Hsp70 expression is the HSE, a series of inverted repeats of the sequence 5Ј-NGAAN-3Ј that bind to oligomeric HSF.…”

“…6D, the Hsp70 promoter containing the GATA site mutation was not induced by p210 BCR-ABL , indicating that this sequence was essential for BCR-ABL-mediated transactivation. Previous work has shown that the major inducible regulator of Hsp70 expression is the HSE, an upstream cis sequence that binds to oligomeric heat shock factor formed in response to elevated temperature (41). To determine whether the BCR-ABL-GATA pathway interacted with the heat shock response-HSE path- way, cells transfected with Ϫ123 Hsp70/LUC in the presence or absence of p210 BCR-ABL were subjected to heat shock.…”

“…Earlier studies have shown that Hsp70 expression is highly inducible at the level of transcription initiation by heat shock and by mitogens acting on a highly inducible heat shock element (HSE) in the proximal Hsp70 promoter (41,42). To identify the major cis regulatory elements responsible for BCR-ABL-driven Hsp70 up-regulation in Phϩ K562 cells, the wild type and a series of 5Ј deletion mutants driving expression of the luciferase reporter were transfected into K562 cells (Fig.…”

Complex Regulation of the Yeast Heat Shock Transcription Factor

“…The promoter of the hsp70 gene of humans, for example, contains binding sites for multiple transcriptional regulators (Morgan et al, 1987;Morgan, 1989). This gene can thus be expressed in response to stress as well as in response to various developmental or other cues.…”

“…E-mail address: jbonner@bio.indiana.edu. † Present address: Parke Davis, Inc., Ann Arbor, MI.© 2000 by The American Society for Cell Biology 1739 (Morgan et al, 1987;Morgan, 1989). This gene can thus be expressed in response to stress as well as in response to various developmental or other cues.…”