Essential Contribution of Intron Sequences to Ca²⁺-Dependent Activation of <i>c-fos</i> Transcription in Pituitary Cells

Abstract

In pituitary cells, c-fos transcription induced by releasing hormones and growth factors results from enhanced initiation of transcription, and sustained elongation of transcripts beyond the first intron. We studied the regulatory role of the first intron of the mouse c-fos gene for the control of its transcription in rat pituitary cells. We showed that the intron contains a block to elongation which is relieved by physiological activators TRH and EGF. By expressing luciferase under the control of the c-fos promoter including the first intron in reporter gene constructs, we demonstrate enhancement of TRH and EGF transcriptional stimulation by intron sequences. Further analysis of Ca²⁺ signalling-depending transcription showed that the intron contains control elements in addition to the block to elongation, and that sequences in the first intron can mediate Ca²⁺-stimulated transcription also with a minimal or the SV40 promoter, irrespective of the presence or absence of the intronic block site. Within the c-fos promoter the serum response element and the cAMP response element play a permissive role in Ca²⁺- and cAMP-enhanced transcription of intron containing reporter genes. Specific binding of nuclear proteins to a consensus enhancer binding site (Sp1) within the first intron of c-fos was demonstrated, which might reflect one of the mechanisms that link Ca²⁺ and intron sequences to c-fos expression. These findings point towards important functions of intronic sequences in gene transcription control.

MAP Kinase Phosphatase-1 Gene Transcription in Rat Neuroendocrine Cells Is Modulated by a Calcium-sensitive Block to Elongation in the First Exon

“…These reporter constructs were compared in basal and stimulated conditions with KCl plus cAMP, TRH, or EGF (Table I). As reported previously (25), expression of luciferase from the c-fos construct is highly induced in all three stimulated conditions, whereas with the MKP-1 promoter, stimulation is limited to a 1.5-1.7-fold increase. Comparing the reporter data from the MKP-1 promoter with the SV40 promoter constructs suggests that the MKP-1 promoter behaves very much like a constitutive promoter in this cell line.…”

“…5). The calcium dependent block to elongation in the intron 1 of c-fos was previously described in neuroendocrine cells (25) and other cell lines (20 -23).…”

“…Plasmid pSV40 was obtained by subcloning the SV40 promoter from the pGL3-control vector (ϩ49 to ϩ244) between the KpnI and NheI restriction sites of the pGL3 enhancer vector. The "c-fos/intron" construction was previously described elsewhere (25,26).…”

“…As a further control, we compared the MKP-1 reporter construct with an SV40 promoter construct and with a "c-fos/ intron" construct that contains the sequence from the promoter to exon 2 fused to the ORF of the luciferase gene (25,26). These reporter constructs were compared in basal and stimulated conditions with KCl plus cAMP, TRH, or EGF (Table I).…”

“…The pMKP-1 construct contains the region Ϫ631 to ϩ19 of the rat MKP-1 gene; pSV40 contains the promoter SV40 of the pGL3-control vector (Promega); and the c-fos/intron construct comprises the region of the mouse c-fos oncogene from the promoter to exon 2 fused to the ORF of the luciferase reporter gene. The transcriptional activity of this construct in GH4C1 was previously described elsewhere (25). DNA fragment.…”