Transcription Factor NFIC Undergoes N-Glycosylation during Early Mammary Gland Involution

Rosemary Kane,Janice Murtagh,Darren Finlay,Andreas Marti,Rolf Jaggi,David Blatchford,Colin Wilde,Finian Martin

doi:10.1074/jbc.m202469200

Abstract

Expression of a 74-kDa nuclear factor I (NFI) protein is triggered in early involution in the mouse mammary gland, and its expression correlates with enhanced occupation of a twin (NFI) binding element in the clusterin promoter, a gene whose transcription is induced at this time (Furlong, E. E., Keon, N. K., Thornton, F. D., Rein, T., and Martin, F. (1996) J. Biol. Chem. 271, 29688-29697). We now identify this 74-kDa NFI as an NFIC isoform based on its interaction in Western analysis with two NFIC-specific antibodies. A transition from the expression of a 49-kDa NFIC in lactation to the expression of the 74-kDa NFIC in early involution is demonstrated. We show that the 74-kDa NFIC binds specifically to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside. In addition, its apparent molecular size was reduced to approximately 63 kDa by treatment with the peptide N-glycosidase. The 49-kDa lactation-associated NFIC did not bind ConA nor was it affected by peptide N-glycosidase. Tunicamycin, a specific inhibitor of N-glycosylation, blocked formation of the 74-kDa NFI in involuting mouse mammary gland in vivo when delivered from implanted Elvax depot pellets. Finally, the production of the ConA binding activity could be reiterated in "mammospheres" formed from primary mouse mammary epithelial cells associated with a laminin-rich extracellular matrix. Synthesis of the 74-kDa NFIC was also inhibited in this setting by tunicamycin. Thus, involution triggers the production of an NFIC isoform that is post-translationally modified by N-glycosylation. We further show, by using quantitative competitive reverse transcriptase-PCR, that there is increased expression of the major mouse mammary NFIC mRNA transcript, mNFIC2, in early involution, suggesting that the involution-associated change in NFIC expression also has a transcriptional contribution.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF358455–AF358460
We show that the 74-kDa NFIC binds to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl ␣-D-mannopyranoside and methyl ␣-D-glucopyranoside
By EMSA (Fig. 1B) we show that the transition from lactation to involution is associated in a change in the dominant species of nuclear factor I (NFI) DNA-binding complex from a relatively smaller (Fig. 1B, lane 5) to a relatively larger species (Fig. 1B, lanes 6 and 7) and that this transition is reversed in the re-differentiated resuckled gland (Fig. 1B, lane 8)

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF358455–AF358460. Associated studies led to the detection of a 74-kDa NFI protein whose expression is triggered in early involution in the mouse mammary gland [40].

Results

Conclusion