Transcription Initiation Is Localized to a TATA-less Region in the Ovine β1 Adrenergic Receptor Gene

doi:10.1006/bbrc.1995.1804

Biochemical and Biophysical Research Communications

Volume 211, Issue 1, 6 June 1995, Pages 254-261

https://doi.org/10.1006/bbrc.1995.1804 Get rights and content

Abstract

We cloned and sequenced the ovine beta₁ adrenergic receptor (β₁AR) gene and promoter region. The transcription start site was localized by RNase protection and primer extension to a GC-rich region. The predominant initiation sequence did not resemble an initiator element and there was no upstream TATA box. Sequence analysis revealed several potential thyroid hormone and glucocorticoid regulatory elements. Identification of the promoter structure of this important gene will help define its novel regulation during development.

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In addition, the increase of [3H]CGP-12177 bindings to both β1- and β2-ARs also occurred simultaneously as demonstrated above. The genes for β1- and β2-ARs of human, rhesus, rat, mouse, and ovine have been identified (Collins et al., 1989, 1993; Shimomura and Terada, 1990; Cohen et al., 1993; Searles et al., 1994, 1995; Padbury et al., 1995; McGraw et al., 1996; Malbon and Hadcock, 1988; Okawa et al., 1998). GC sequences are rich in the early promoter regions of both genes and a consensus TATA box is lacking.
Mechanisms of up-regulation of β-adrenergic receptors (β-ARs) induced by sustained exposure to 10^− 8 M nadolol, a non-selective β-AR antagonist, were examined using mouse cerebrocortical neurons. Nadolol dose- and time-dependently increased [³H]CGP-12177 bindings to the particulate fractions. This increase occurred 6 h and attained its plateau 12 h after the exposure, whereas β₁- and β₂-AR mRNA significantly increased 24 h and attained their plateaus 3 days after the exposure. Scatchard analysis revealed that the increased bindings were due to increase of receptor density. The [³H]CGP-12177 bindings to β₁- and β₂-ARs increased both 12 h and 5 days after the exposure. Although cycloheximide (CHX) decreased the bindings with or without nadolol, the extent of increase of the bindings induced by nadolol was not affected by CHX. Actinomycin D (AD) with nadolol showed no affects on the bindings 12 h after nadolol exposure, while AD treated 6 h after nadolol exposure significantly reduced the bindings 48 h after nadolol exposure. During 24 h after nadolol exposure, the increase in proteins of β₁- and β₂-ARs in the neuronal membrane was due to the increased receptor protein translocation from cytosol to membrane. These results indicate that the up-regulation of β-ARs induced by nadolol is mediated by, at least, two different processes, one is increase in translocation of receptor proteins from cytosol to membrane with no changes in synthesis of receptor proteins and their mRNA and another is dependent on receptor protein synthesis with increased synthesis of their mRNA.
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The effects of glucocorticoids on expression of the β1-adrenergic receptor (β1AR) gene have been varied. To study the mechanism underling hormonal regulation of the β1AR, transient transfection of progressively deleted ovine β1AR promoter fragments was used to identify a 43-bp region (−1274 to −1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) and confers glucocorticoid responsiveness. Using DNase I footprinting and electrophoretic mobility shift assays (EMSA), we demonstrated the GRU was composed of a palindrome, 5′-TAATTA-3′, which is a core binding motif for the homeodomain proteins, an E-box (5′-CACGTG-3′), binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element (GRE) half-site (5′-TGTTCT-3′). EMSA demonstrated that the GRE half-site is critical for GRU–protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Co-transfection of a plasmid expressing a c-myc antisense construct significantly reduced glucocorticoid responsiveness of the ovine β1AR promoter. Furthermore, expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic, reduced in newborn and not detectable in adult heart. We conclude that the ovine β1AR promoter contains a novel, functional GRU and that glucocorticoid receptor (GR) and the Myc/Max family proteins are involved in the cell-specific nuclear factor binding and transactivation via this element. The results suggest an alternative pathway through which glucocorticoids may exert their effects on genes lacking a full consensus GRE.
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Rat β1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins
2000, Molecular Cell Biology Research Communications
β1-Adrenergic receptors (β1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using β1-AR-luciferase reporter recombinants, we have previously determined that important β1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region −396 to −299, relative to the translational start site. By conducting progressive internal deletions of the rat β1-AR 5′ flanking region and with the use of β1-AR-luciferase recombinants, we have verified that this region contains the primary β1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in β1-AR transcriptional activity conferred by this β1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined β1-AR DNA subregion probes. One probe (GS-1), encompassing the region −396 to −367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the β1-AR expresser C6 cell line. UV-crosslinking of DNA–protein complexes, coupled with DNase I digestion, indicated that this β1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA–protein complexes were observed using β1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA–protein complexes were observed when using nuclear extracts from β1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no β1-ARs, a reduction in intensities of the DNA–protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein will bind to both the β1-AR GS-1 promoter fragment and commercially available AP-2 consensus element control probes. Interestingly, using antibody supershift and immunoblotting experiments, no supershifts were observed and the major 117-kDa protein was not immunoreactive to antibodies recognizing either AP-2α or AP-2β. These results support our contention that this β1-AR regulatory region contains AP-2 consensus elements that recognize novel transactivator proteins.

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Regular ArticleTranscription Initiation Is Localized to a TATA-less Region in the Ovine β1 Adrenergic Receptor Gene

Abstract

Regular Article
Transcription Initiation Is Localized to a TATA-less Region in the Ovine β1 Adrenergic Receptor Gene