DNA AND CELL BIOLOGY Volume 11, Number 1, 1992 Mary Ann Lieber!, Inc., Publishers Pp. 61-69

The Cloned Promoter of the Human DNA ß-Polymerase Gene Contains a cAMP Response Element Functional in HeLa Cells ELLA W. ENGLANDER and SAMUEL H. WILSON

ABSTRACT The mammalian DNA /3-polymerase (/3-pol) gene is constitutively expressed in cultured cells as a function of growth stage and DNA replication, but is expressed in rodents in a tissue-specific fashion. As revealed by transient expression experiments with wild-type and mutated /3-pol promoter fusion genes, the cloned human j-pol promoter is transcriptionally regulated by signals acting through the single palindromic sequence (GTGACGTCAC) known as an ATF/CRE-binding site centered at position -45 in the core promoter. Although the mere presence of the ATF/CRE palindromic sequence in a promoter does not always confer cAMP responsiveness or protein binding over and around the ATF/CRE sequence, we find that agents that increase cAMP levels (forskolin and IBMX) in HeLa cells activate the /3-pol promoter; activation also can be observed by coexpression of the protein kinase A catalytic subunit. Experiments with mutagenized /3-pol promoters indicate that the ATF/CRE-binding site mediates these effects. Thus, the ATF/CRE-binding site in the context of this TATA-less constitutive promoter is able to respond to the kinase A signal transduction

pathway.

INTRODUCTION cellular (/3-pol) polymerizDNAing /3-polymerase (Fry Loeb, 1986; its the cell

regulate the promoter through this element. This element at position -40 to -49 in the core promoter (Widen et ai, 1988), forms the center of a strong DNase I footprinting site for nuclear extract proteins from a variety of tissues and cell lines (Englander and Wilson,

N'-nitro-N-nitrosoguanidine (MNNG) (Kedar et ai, 1991). Hence, the diversity of signals mediated through the ATF/ CRE site suggests that several signal transduction path-

conferred at this site by the extracts from a variety of tissues and cells; however, corresponding gel retardation assays revealed tissue- and cell line-specific bandshift patterns, consistent with the existence of several proteins binding to the /3-pol promoter ATF/CRE site (Englander and Wilson, 1990). One of these DNA-binding protein species purified to near-homogeneity from bovine testes is a phosphoprotein with multiple phosphorylation sites in vivo and a strong in vitro substrate for protein kinase A (Englander et ai, 1991). Many genes that are transcriptionally activated by cAMP share in their 5'-flanking region a conserved palindromic motif corresponding to the sequence TGACGTCA (for review, see Deutsch et ai, 1988; Roesler et ai, 1988; Ziff, 1990). This element in several promoters binds pro-

ways may converge to

is a DNA and Wilenzymes in vertebrates son et ai, 1988); role in concerns short gap-filling synthesis during DNA repair (Fry and Loeb, 1986; Hammond et ai, 1990; Wiebauer and Jiricny, 1990). 0-pol is a constitutive "housekeeping enzyme," yet its expression in rodents varies in a tissue-specific manner (Zmudzka et ai, 1988; Hirose et ai, 1989; Nowak et ai, 1990). Functional analyses of a human /3-pol promoter fusion gene by transient expression experiments show that integrity of the single ATF/CRE palindromic site in the core promoter (GTGACGTCAC) is essential for full promoter activity and the /raws-activation observed by E1A/E1B (Widen et ai, 1988), for the stimulation observed with activated p2K

The cloned promoter of the human DNA beta-polymerase gene contains a cAMP response element functional in HeLa cells.

The mammalian DNA beta-polymerase (beta-pol) gene is constitutively expressed in cultured cells as a function of growth stage and DNA replication, but...
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