Identification of a silencer module which selectively represses cyclic AMP-responsive element-dependent gene expression.
The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.
Chung
K C
KC
Department of Cellular and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Huang
D
D
Chen
Y
Y
Short
S
S
Short
M L
ML
Zhang
Z
Z
Jungmann
R A
RA
D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000502:physiology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D018983:DNA Footprinting; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics*; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D012097:Repressor Proteins; D000242:Cyclic AMP; D007770:L-Lactate Dehydrogenase