Abstract
Treatment of cells with the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase beta (beta-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG-induced up-regulation. Recently, we described a kinetic mechanism for transcriptional regulation of the beta-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up-regulates the promoter by stimulating formation of closed preinitiation complex (RPc). Here, using a CRE-dependent chimeric beta-pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RPc samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the beta-pol promoter after treatment of cells with the DNA-alkylating agent MNNG.
Highlights
The same nuclear extract (NE) supplemented with 10 ng of unphosphorylated CRE-binding protein (CREB)-1 showed promoter activity that was the same as control NE alone. These results are consistent with the idea that phosphorylated CREB-1 is limiting in the control cell NE and that phosphorylation of CREB-1 is stimulated by MNNG treatment, in turn resulting in upregulation of the -pol promoter activity
The present study describes characterization of a CREB family member that is involved in -pol gene regulation, as well as a mechanism for transcriptional activation of -pol gene expression by an alkylating agent in vivo
With the cloned human -pol promoter, indicated that the PKA signal transduction pathway plays a required role in transcriptional activation after exposure of cells to MNNG [12, 13, 15] and that the cAMP response element (CRE) site of the -pol promoter is required for the MNNG response [15]
Summary
Using a CRE-dependent chimeric -pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the -pol promoter after treatment of cells with the DNA-alkylating agent MNNG. In the present study, using purified RPc assembled from NE of control and MNNG-treated cells, experiments were performed to identify and characterize the RPc-associated CREB.
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