The palindromic series i repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements

Yung Nien Chang, Simon Crawford, Jacintha Stall, Dan R. Rawlins, Kuan Teh Jeang, Gary Selwyn Hayward

Research output: Contribution to journalArticle

Abstract

A 600-base-pair (bp) enhancer region upstream from the major IE94 gene of simian cytontegalovirus (SCMV) produces very strong basal expression of associated gene products. This domain consists of multiple sets of interspersed repetitive elements, including 11 copies of a conserved 16-bp palindromic sequence with the consensus CCATTGACGTCAATGG. These series I repeats contain an 8-bp core TGACGTCA that resembles the cyclic AMP (cAMP) response element (CRE) of cellular genes. In transient chloramphenicol acetyltransferase assays in K562 human erythroleukemia cells, a set of deleted variants of the IE94 promoter all responded up to 15-fold to induction by cAMP. However, successive removal of most of the SCMV 16-bp motifs reduced basal expression over 20-fold. The cAMP stimulation was also manifested at the steady-state RNA level after SCMV infection of K562 cells and was detectable within 1.5 h after treatment of DNA-transfected cells. Addition of a single 30-bp oligonucleotide encompassing the 16-bp palindrome conveyed up to 10-fold cAMP responsiveness onto a heterologous weak promoter but had no effect on basal expression. In contrast, two or more adjacent copies produced 20- to 40-fold increases in basal expression and provided greater than 200-fold activation in the presence of cAMP. Similar effects were obtained when the oligonucleotides were placed in a downstream location relative to the reporter gene. Studies with mutant oligonucleotides revealed that both the core CRE and the flanking sequence portions of the 16-bp elements were essential for enhancer function. Both components were also important for maximum cAMP responsiveness. Band shift assays with fractionated nuclear extracts from Raji lymphocytes revealed multiple competable complexes with cellular DNA-binding factors that recognized the series I elements. Three distinct CREB-like factors were detected that required only the core 8-bp elements for binding. We conclude that the 16-bp series I repeats provide a major contribution to the constitutive enhancer properties of the IE94 promoter and also act as functional CREs. The cAMP response properties appear likely to play a key role in reactivation of the virus from a latent state in appropriately differentiating cell types.

Original languageEnglish (US)
Pages (from-to)264-277
Number of pages14
JournalJournal of Virology
Volume64
Issue number1
StatePublished - 1990

Fingerprint

Cytomegalovirus
response elements
cyclic AMP
Response Elements
Base Pairing
Cyclic AMP
promoter regions
oligonucleotides
Oligonucleotides
cells
chloramphenicol acetyltransferase
Interspersed Repetitive Sequences
DNA
assays
major genes
reporter genes
Leukemia, Erythroblastic, Acute
Chloramphenicol O-Acetyltransferase
K562 Cells
Consensus Sequence

ASJC Scopus subject areas

  • Immunology

Cite this

The palindromic series i repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements. / Chang, Yung Nien; Crawford, Simon; Stall, Jacintha; Rawlins, Dan R.; Jeang, Kuan Teh; Hayward, Gary Selwyn.

In: Journal of Virology, Vol. 64, No. 1, 1990, p. 264-277.

Research output: Contribution to journalArticle

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abstract = "A 600-base-pair (bp) enhancer region upstream from the major IE94 gene of simian cytontegalovirus (SCMV) produces very strong basal expression of associated gene products. This domain consists of multiple sets of interspersed repetitive elements, including 11 copies of a conserved 16-bp palindromic sequence with the consensus CCATTGACGTCAATGG. These series I repeats contain an 8-bp core TGACGTCA that resembles the cyclic AMP (cAMP) response element (CRE) of cellular genes. In transient chloramphenicol acetyltransferase assays in K562 human erythroleukemia cells, a set of deleted variants of the IE94 promoter all responded up to 15-fold to induction by cAMP. However, successive removal of most of the SCMV 16-bp motifs reduced basal expression over 20-fold. The cAMP stimulation was also manifested at the steady-state RNA level after SCMV infection of K562 cells and was detectable within 1.5 h after treatment of DNA-transfected cells. Addition of a single 30-bp oligonucleotide encompassing the 16-bp palindrome conveyed up to 10-fold cAMP responsiveness onto a heterologous weak promoter but had no effect on basal expression. In contrast, two or more adjacent copies produced 20- to 40-fold increases in basal expression and provided greater than 200-fold activation in the presence of cAMP. Similar effects were obtained when the oligonucleotides were placed in a downstream location relative to the reporter gene. Studies with mutant oligonucleotides revealed that both the core CRE and the flanking sequence portions of the 16-bp elements were essential for enhancer function. Both components were also important for maximum cAMP responsiveness. Band shift assays with fractionated nuclear extracts from Raji lymphocytes revealed multiple competable complexes with cellular DNA-binding factors that recognized the series I elements. Three distinct CREB-like factors were detected that required only the core 8-bp elements for binding. We conclude that the 16-bp series I repeats provide a major contribution to the constitutive enhancer properties of the IE94 promoter and also act as functional CREs. The cAMP response properties appear likely to play a key role in reactivation of the virus from a latent state in appropriately differentiating cell types.",
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