Functional analysis of the mouse ICER (Inducible cAMP Early Repressor) promoter: evidence for a protein that blocks calcium responsiveness of the CAREs (cAMP autoregulatory elements).
Although Ca2+ and cAMP mediate their effects through distinct pathways, both signals converge upon the phosphorylation of the cAMP response element (CRE) binding protein, CREB, thereby activating transcription of CRE-regulated genes. In WEHI7.2 thymocytes, cAMP increases the expression of the inducible cAMP early repressor (ICER) gene through CRE-like elements, known as cAMP autoregulatory elements (CAREs). Because Ca2+ -and cAMP-mediated transcription converge in WEHI7.2 thymocytes, we examined the effect of Ca2+ fluxes on the expression of the ICER gene in these cells. Despite the presence of multiple CAREs within its promoter, ICER gene transcription was not activated by Ca2+. Moreover, Ca2+ attenuated the stimulatory effect of cAMP on ICER expression. Transient expression of reporter constructs demonstrated that when these CAREs were placed in a different DNA promoter context, the elements became responsive to Ca2+. Detailed studies using chimeric promoter constructs to map the region responsible for blocking the transcriptional response to Ca2+ indicated that a small portion of the ICER promoter was necessary for the effect. Southwestern blot analysis identified a 83-kDa nuclear protein that bound specifically to that region. The relative binding activity of the factor to the ICER promoter and mutant promoter sequences correlated with an inhibition of Ca2+ -activated gene expression in WEHI7.2 cells. These data suggest that the factor functions as a putative Ca2+ -activated repressor of CREB/CRE-mediated transcription. Thus, depending on the surrounding context in which the CRE is located, CREs of individual genes can be regulated separately by Ca2+ and cAMP despite the convergence of these two signaling pathways.
E. A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, Missouri 63104, USA.
D A
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Krueger
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D
Mao
E A
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Warner
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Dowd
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D051786:Cyclic AMP Response Element Modulator; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism; D012097:Repressor Proteins; D020218:Response Elements / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D011506:Proteins; D012097:Repressor Proteins; D051786:Cyclic AMP Response Element Modulator; D000242:Cyclic AMP; D002118:Calcium