Nuclear Factor I-mediated Repression of the Mouse Mammary Tumor Virus Promoter Is Abrogated by the Coactivators p300/CBP and SRC-1

Ali Z Chaudhry,Alfredo D Vitullo,Richard M Gronostajski

doi:10.1074/jbc.274.11.7072

Ali Z Chaudhry, Alfredo D Vitullo + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.11.7072

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Abstract

To better understand the function of nuclear factor I (NFI) proteins in transcription, we have used transient transfection assays to assess transcriptional modulation by NFI proteins on the NFI-dependent mouse mammary tumor virus (MMTV) promoter. Expression of NFI-C or NFI-X, but not NFI-A or NFI-B proteins, represses glucocorticoid induction of the MMTV promoter in HeLa cells. Repression is DNA binding-independent as a deletion construct expressing the NH2-terminal 160 residues of NFI-C represses but does not bind DNA. Repression by NFI-C is cell type-dependent and occurs in HeLa and COS-1 cells but not 293 or JEG-3 cells. NFI-C does not repress progesterone induction of the MMTV promoter in HeLa cells, suggesting that progesterone induction of the promoter differs mechanistically from glucocorticoid induction. NFI-C-mediated repression is alleviated by overexpression of glucocorticoid receptor (GR), suggesting that NFI-C represses the MMTV promoter by preventing GR function. However, repression by NFI-C occurs with only a subset of glucocorticoid-responsive promoters, as the chimeric NFIGREbeta-gal promoter that is activated by GR is not repressed by NFI-C. Since the coactivator proteins p300/CBP, SRC-1A, and RAC3 had previously been shown to function at steroid hormone-responsive promoters, we asked whether they could influence NFI-C-mediated repression of MMTV expression. Expression of p300/CBP or SRC-1A alleviates repression by NFI-C, whereas RAC3 has no effect. This abrogation of NFI-C-mediated repression by p300/CBP and SRC-1A suggests that repression by NFI-C may occur by interference with coactivator function at the MMTV promoter.

Highlights

Cloning of cDNAs encoding nuclear factor I (NFI) proteins from several species [13,14,15] has identified a family of four genes (NFI-A, NFI-B, NFI-C, and NFI-X) that are highly conserved from chickens to humans
Our previous studies showed that NFI proteins representing each of the four NFI genes differentially activated the mammary tumor virus (MMTV) promoter in human JEG-3 choriocarcinoma cells, with NFI-B activating expression ϳ12-fold, NFI-X activating ϳ10-fold, NFI-C ϳ6-fold, and NFI-A only ϳ2-fold
Repression by NFI-C is overcome by overexpression of the glucocorticoid receptor (Fig. 3A), suggesting that repression may occur through interference with GR function

Summary

Introduction

Cloning of cDNAs encoding NFI proteins from several species [13,14,15] has identified a family of four genes (NFI-A, NFI-B, NFI-C, and NFI-X) that are highly conserved from chickens to humans. Direct interactions between a human NFI-C isoform (CTF1) and components of the basal transcriptional machinery have been reported, with interactions dependent on a sequence motif related to the COOH-terminal heptapeptide repeat (CTD) of RNA polymerase II [23]. This is unlikely to be the only mechanism by which NFI proteins activate transcription as some NFI proteins lacking a CTD repeat are potent activators in both yeast [24] and mammalian cells [21]. We show that NFI proteins exhibit cell type- and promoter-specific differences in their repression properties, with NFI-C and -X repressing the MMTV promoter response element; PBS, phosphate-buffered saline; h, human; aa, amino acids. We demonstrate that overexpression of p300/CBP or SRC-1A abrogates repression of MMTV expression, suggesting that NFI-

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