Gastroprotective peptide trefoil factor family 2 gene is activated by upstream stimulating factor but not by c-Myc in gastrointestinal cancer cells

Abstract

Background: Damage to the gastrointestinal mucosa results in the acute up-regulation of the trefoil factor family peptides TFF1, TFF2, and TFF3. They possess protective, healing, and tumour suppressive functions. Little is known about the regulation of TFF gene expression. The promoters of all three TFF genes contain binding sites (E box) for upstream stimulating factor (USF) and Myc/Max/Mad network proteins.

Aims: To determine the nature and function of transcription factors that bind to these E boxes and to understand their role for TFF gene expression.

Methods: TFF promoter activities were determined by reporter gene assays. DNA binding was monitored by electromobility shift assays and by chromatin immunoprecipitation analyses. Expression of endogenous TFF was determined by multiplex RT-PCR.

Results: It was observed that the TFF2 promoter is specifically and efficiently activated by USF transcription factors but not by c-Myc. USF displayed comparable binding to a high affinity Myc/Max binding site compared with the three TFF E boxes, while c-Myc exhibited lower affinity to the TFF E boxes. In contrast, pronounced binding differences were observed in cells with a strong preference for USF to interact specifically with the TFF2 E box, while Myc was not above background. Exogenous expression of USF was sufficient to activate the chromosomal TFF2 and to a lesser extent, the TFF1 gene.

Conclusion: These findings define USF factors as regulators of the TFF2 gene and suggest that promoter specific effects are important for a pronounced gene activation of this cytoprotective peptide.

Figure 1

Schematic representation of the promoters of the three TFF genes. The locations and sequences (deviation from the consensus are underlined) of E box elements in relation to the core promoters are given. Binding sites for C/EBP factors (C), HNF-3 (H), and GATA-6 (G) are indicated. The TATA box in TFF3 is CATAAA.

Figure 2

USF factors activate a TFF2-luc reporter gene construct. (A) The gastrointestinal cell line MKN45 was cotransfected with the TFF2-luc reporter gene construct and indicated amounts of expression plasmids encoding USF1, USF2, USF1/USF2, or c-Myc. (B) As in (A) but the cell line LS174T was used. (C) MKN45 cells were cotransfected with wild type (wt) or E box mutated (mut) TFF2-luc, with (+) or without (-) expression plasmids encoding USF1 or a mutant USF protein that cannot bind DNA (m). (D) As in (A) but the M4-mintk-luc reporter gene construct with four high affinity Myc/Max binding sites was used. (E) As in B but with the M4-mintk-luc reporter gene.

Figure 3

Effects of USF1 on TFF1-luc and TFF3-luc. MKN45, LS174T, and HT-29 cells were cotransfected with the indicated TFF reporter gene constructs in the presence or absence of USF1. The fold induction by USF1 is shown.

Figure 4

Binding of USF, c-Myc/Max, and Max/Max complexes to TFF derived E box sequences. (A) Binding of COS-7 derived USF1/USF1 homodimers to radiolabelled CMD was competed with a 3-fold, 10-fold, 30-fold, or 100-fold excess of unlabelled CMD, or oligonucleotides spanning the E boxes of either TFF1, TFF2, or TFF3 as indicated. Supershift experiments were performed with an antibody specific for USF1 (α-USF1). Control: extract of mock transfected cells was used. * Non-specific complex. (B) Binding of COS-7 derived c-Myc/Max and Max/Max complexes were analysed as in (A) Supershift experiments were done with specific antibodies to c-Myc or Max (α-Myc or α-Max, respectively). The control is as in (A). * Non-specific complex.

Figure 5

Binding of USF to the TFF2 promoter in cells. (A) ChIP analyses were performed on lysates of formaldehyde crosslinked HT-29 and MKN45 cells. One of 20 of the purified DNA from individual immunoprecipitations was subjected to PCR analysis (34 cycles). Fourfold dilution series of purified input DNA served as normalisation controls for the PCR reactions. (B) Quantification of ChIP reactions from HT-29 cells. Product intensities of samples and adequate twofold dilution series of purified input DNA were compared. Relative DNA binding defines the amount of PCR product obtained from immunoprecipitates compared with total input DNA with the amount of PCR product equivalent to 80 pg arbitrarily set as 1. The graph shows the mean value of two independent determinations. Insert: Lower amounts of input DNA were used to determine c-Myc and c-Myb binding equivalents (39 cycles). (C) ChIP analyses were performed as described under (A).

Figure 6

Exogenous USF stimulates the expression of the chromosomal copy of TFF2. LS174T cells were transiently transfected with empty vector or with plasmids encoding USF1 and USF2 or c-Myc as indicated. Endogenous TFF mRNA expression was analysed after 24 cycles of multiplex RT-PCR. Lanes 1: TFF1-PCR, GAPDH-PCR, TFF3-PCR reactions; lanes 2: TFF1-PCR, GAPDH-PCR, TFF2-PCR reactions.