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

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 ␣-D-mannopyranoside and methyl ␣-D-glucopyranoside. In addition, its apparent molecular size was reduced to ϳ63 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.

The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

“…NFIC also has been reported previously to be modified by N-glycosylation [5]. Kane et al have reported that a 74 kDa N-glycosylated NFIC isoform appears during mammary gland involution [9]. But the specific role of this N-glycosylated isoform is not well understood.…”

“…However, we have shown previously that the NFI-B2 isoform displayed an increased binding affinity as compared to the NFI-X1 and NFI-A4 isoforms to the WAP promoter. Subsequent studies by other investigators have also revealed the differential binding affinity of specific NFI isoforms to their DNA binding motifs in other promoters [9]. While these differences in DNA binding affinity may help facilitate the specific competition of one NFI isoform with other NFI proteins, these differences alone may not be sufficient to account for their selective ability to cooperate with the other transcription factors for the regulation of the mammary-specific genes.…”

“…This region is highly proline-rich and also contains several serine and threonine residues as potential targets for either phosphorylation and/or O-glycosylation [5]. An involution-associated 74kDa NFI-C isoform has been identified which is post-translationaly modified by N-glycosylation [9,23,24]. We, therefore, compared the post-translation modification between the C-terminal regions of both the NFI-A4 and B2 to determine why the C-terminal region of NFI-B2, but not of A4 can activate the WAP CoRE in the JEG cells.…”

“…Furthermore, a mammary cell-specific enhancer in the mouse mammary tumor virus (MMTV) promoter includes an active NFI binding site [8]. In addition, the involutionassociated transcription of the TRPM-2/clusterin gene has been shown to be controlled by a unique N-glycosylated 74 kDa NFI-C isoform [9].…”

N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H

“…Glycosylation is a major form of posttranslational modification and its regulatory roles in the context of different transcription factors such as CREB, NF1C, Oct-2, Sp1 and STAT5 have been documented (Jackson and Tjian, 1988;Kane et al, 2002;LamarreVincent and Hsieh-Wilson, 2003;Gewinner et al, 2004;Ahmad et al, 2006). In addition, ATF6, another membrane-bound bZIP transcription factor activated by RIP in response to ER stress, is also known to be N-linked glycosylated and this modification has an impact on its transcriptional activity (Hong et al, 2004).…”