Mammary Gland - Transcription Factor NF 1 Expression in Involuting Mammary Gland

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Transcription Factor

Transcription Factor NF 1 Expression in Involuting Mammary Gland

1. INTRODUCTION

       Clusterin gene expression is rapidly induced in early involution of the mouse mammary gland after weaning. A search for involution enhanced DNaseI footprints in the proximal mouse TRPM-2/clusterin gene promoter led to the identification and characterization (by DNase I footprinting) of a twin NF1 binding element at -356/-309, relative to the proposed transcription start-site, with nuclear extracts from 2d-involuting mouse mammary gland showing an enhanced footprint over the proximal NF1 element, relative to extracts from lactating mammary gland. Subsequent EMSA and western analysis led to the detection of a 74 kD NF1 protein whose expression is triggered in early involution in the mouse mammary gland. This protein was not found in lactation where three other NF1 proteins of 114, 68 and 46 kD were detected. Reiteration of the epithelial cell apoptosis associated with early mammary gland involution, in vitro, in a primary cell culture system, triggered the appearance of the 74 kD NF1. Overlaying the cells with laminin-rich ECM suppressed the apoptosis and the expression of the 74 kD NF1 and, in the presence of lactogenic hormones, initiated milk protein gene expression and the expression of two of the lactation associated NF 1 proteins (68 and 46 kD). Below we describe the transcripts of NF1 genes in mouse mammary gland which potentially encode the involution-associated 74 kD NF1.

2.  MATERIALS AND METHODS

     The basic procedures employed are described by Furlong et al. . Specific PCR analyses employed hot start and suitable cycling conditions established for each primer pair.

3. RESULTS
       NF1 (nuclear factor 1) and CTF/NF1 (CCAAT-binding transcription factors/NF 1) proteins are a heterogeneous family of transcription factors which have a highly conserved N-terminal DNA binding and dimerisation domain and proline-rich carboxy-terminal transactivating domains that are highly divergent in size and sequence. Thus, related products encoded by four different genes have been characterized NF1-A, B, C and X) and multiple splice variants which differ in the carboxy terminus of these have been described. Based on the sequence of known transcripts of the four mouse NF-1 genes we devised an RT-PCR strategy that would allow us clone the NF1 transcripts expressed in lactating and involuting mouse mammary gland. Table 1 shows that multiple transcripts of each of the four NF1 genes (NF1 A, B, C (CTF/NF1) and X) are expressed in both lactating and involuting mouse mammary gland. Three transcripts of NF1 A, two of NF1 B and two of NF1 X were cloned and characterised (by DNA sequencing) from both lactating and early involuting mammary gland. Only in the case of NF1 C (CTFNF1) were transcripts found exclusively in either lactating or involuting mammary tissue. However, these were seemingly rare and previously undescribed transcripts: NF1 C-∆-5^8,9,10* was detected in lactating tissue; and, NF1 C-∆-8,9 and NF1 C-∆-5^8,9 were detected in involuting tissue (numbers denote the spliced-out exons; 5^8, spliced from within exon 5 to within exon 8). Table I.: NF1 Transcripts Expressed in the Mouse Mammary Gland

Transcript	Lactation	Involution
NF1A
mNF1-A1 (Y07690*)	+	+
mNF1-A2 (Y07691)	+	+
NF1-B
mNF1-B1 (Y07685)	+	+
mNF1-B2 (Y07687)	+	+
mNF1-B3 (Y07686)	+	+
NF1-X
mNF1-X1 (Y07688)	+	+
mNF1-X2 (Y07689)	+	+
mNF1-X3 (S81451)	-	-
NF1-C
(h)CTF- 1 (X 12492)	-	-
mNF1-C2, (U57635, Y07692, Y07693)	+	+
mCTF-5 (X92857)**	+	+
Novel mouse NF1-C
mNF1-C3 (Exons 7&9 spliced)	+	+
mNF1-C4 (Exons 8&9 spliced)	-	+
mNFl-C4 (Exons 7&9 spliced)**	+	+
mNF1-C6 (Exons 5^8&9 spliced)	-+
mNF1-C7 (Exons 5"8,9&10 spliced)	+	-

*Genbank accession numbers are provided for all previously cloned NF1s **Described previously for human not for mouse
We next designed specific primer sets for PCR analysis of total NF1 A, NF1 B and NF1 X transcripts and the predominant individual NF1 A, B and X transcripts in lactating (L), involuting (2 di) and re-suckled 2-day involuting mammary gland (36h s). There was no striking difference in total NF1 A, B or C transcript levels between lactating, involuting or re-differentiated mammary tissue; however, analysis of the individual NF1 A transcripts suggested that there was greater expression of NF1 A1 in lactation and the resuckled gland than in involution (Fig 1). In the case of the NF1 B family members a bias towards increased expression in involution was seen; no discernible differences could be established for the individual NF1 X transcripts (Fig. 1). While these experiments suggested that the involution-associated 74 kD NF1 was possibly encoded by an NF1 B gene we obtained more compelling evidence that it was encoded by an NF1 C/CTF-NF1 gene: The 74kD involution-associated-NF1 was detectable in western analysis by a range of antibodies raised against the conserved N-terminal NF1 DNA binding domain but also by an anti-CTF/NF1 C-specific antibody. We established that this latter antibody is seemingly NF1 C-specific by its ability to detect HA-epitope-tagged mouse NF1 C2 transiently expressed in JEG 3 cells (which have low endogenous NF1 expression ; the antibody did not detect expressed epitope-tagged NF1 A, B or X. An anti-HA antibody detected all four expressed proteins in transiently transfected JEG 3 cells (results not shown).

Figure 1. NF1 transcript levels in lactating and involuting mouse mammary gland

The dominant NF1 C transcripts detectable in mammary epithelial cells are NF1 C2 and NF1 C5. We established an RT PCR assay for these two transcripts (Fig. 2). We found a definite tendency towards higher expression of these transcripts in involuting mammary gland than in lactating and re-differentiated mammary gland (Fig. 2). However, this increased NF1 C transcript expression tends to be higher on day 3 of involution while expression of the 74 kD involution-associated protein tends to be higher on day 2 of involution.

4. CONCLUSION

The involution-associated 74 kD NF1 (and the 114 kD lactation-associated NF1 are recognised by an anti-NF1 C–specific antibody (see above). It is most likely, therefore, that this lactation/involution switch in NF 1 factors represents a change in expression of NF1 C (CTF/NF1) proteins. We present preliminary evidence here that there is also an involution-associated increase in NF1 C transcript levels but highest levels are detectable slightly later in involution (day 3), after highest levels of the 74 kD protein are seen (day 2). Of significant consequence, also, is the fact that the known mouse NF1 C transcripts encode proteins of approximately < 60 kD. Thus, the 74 kD NF 1 is a post-translationally modified NF1 C or encoded by an NF1 transcript of novel exonic structure. We are presently endeavouring to answer these questions.

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