Basal Expression of IκBα Is Controlled by the Mammalian Transcriptional Repressor RBP-J (CBF1) and Its Activator Notch1

Fiona Oakley,Jelena Mann,Richard G Ruddell,Jessica Pickford,Gerry Weinmaster,Derek A Mann

doi:10.1074/jbc.m211051200

Abstract

By using the hepatic stellate cell (HSC) as a paradigm for cells that undergo long term re-programming of NF-kappaB-dependent transcription, we have determined a novel mechanism by which mammalian cells establish their basal NF-kappaB activity. Elevation of NF-kappaB activity during HSC activation is accompanied by induction of CBF1 expression and DNA binding activity. We show that the transcriptional repressor CBF1 interacts with a dual NF-kappaB/CBF1-binding site (kappaB2) in the IkappaBalpha promoter. Nucleotide substitutions that disrupt CBF1 binding to the kappaB2 site result in an elevation of IkappaBalpha promoter activity and loss of responsiveness of the promoter to a transfected CBF1 reporter vector. Overexpression of CBF1 in COS1 cells was associated with markedly reduced IkappaBalpha protein expression and elevated NF-kappaB DNA binding activity. CBF1-induced repression of IkappaBalpha promoter activity was reversed in HSC transfected with the Notch1 intracellular domain (NICD). The ability of NICD to enhance IkappaBalpha gene transcription was confirmed in COS1 cells and was found to be dependent on an intact RAM domain of NICD that has been shown previously to help mediate the interaction of NICD with CBF1. One of the mechanisms by which NICD is thought to convert CBF1 into an activator of transcription is via the recruitment of transcriptional co-activators/histone acetylases to gene promoters. Co-transfection of HSC with NICD and p53 caused a diminution of IkappaBalpha promoter activity, by contrast overexpression of p300 enhanced IkappaBalpha promoter function. Taken together, these data suggest that basal IkappaBalpha expression (and as a consequence NF-kappaB activity) is under the control of the various components of the CBF1/Notch signal transduction pathway.

Highlights

The transcription factor NF-␬B is a key regulator of the growth, differentiation, and fate of mammalian cells [1]
hepatic stellate cell (HSC) cultured for 7 and 10 days expressed high levels of a protein species that was recognized by the CBF1 antibody and that was of a similar size (57 kDa) to CBF1 expressed in COS1 cells transfected with the CBF1 expression vector pJH282 (Fig. 2A)
CBF1 mRNA expression was carried out on RNA isolated from various time points of culture activation of HSC using primers based on the murine CBF1 cDNA sequence predicted to generate a product of 473 bp

Summary

Introduction

The transcription factor NF-␬B is a key regulator of the growth, differentiation, and fate of mammalian cells [1]. The three most important I␬B proteins, I␬B␣, I␬B␤, and I␬B⑀, inhibit NF-␬B function by masking nuclear localization and DNA binding signals [1, 3] These inhibitors are pivotal regulators of NF-␬Bdependent gene expression as they can be degraded in response to a wide variety of epigenetic stimulators and in doing so release active NF-␬B which can translocate to the nucleus and activate transcription [3]. These cells have been reported to exhibit reduced expression of I␬B␣, which either results from amino acid mutations that reduce stability of the I␬B␣ protein or from higher constitutive I␬B kinase activity (28 –30) Despite these examples, little is understood about the signaling mechanisms that regulate basal NF-␬B activity in the majority of differentiated mammalian cells

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Conclusion