The promoter for intestinal cell kinase is head-to-head with F-Box 9 and contains functional sites for TCF7L2 and FOXA factors

Abstract

Background: Intestinal cell kinase (ICK; GeneID 22858) is a conserved MAPK and CDK-like kinase that is widely expressed in human tissues. Data from the Cancer Genome Anatomy Project indicated ICK mRNA is increased in cancer, and that its expression correlated with expression of mRNA for an uncharacterized F-box protein, FBX9 (GeneID: 26268). ICK and FBX9 genes are arranged head-to-head on opposite strands, with start sites for transcription separated by approximately 3.3 kb. We hypothesized ICK and FBX9 are potentially important genes in cancer controlled by a bidirectional promoter.

Results: We assessed promoter activity of the intergenic region in both orientations in cancer cell lines derived from breast (AU565, SKBR3), colon (HCT-15, KM12), and stomach (AGS) cancers, as well as in embryonic human kidney (HEK293T) cells. The intergenic segment was active in both orientations in all of these lines, and ICK promoter activity was greater than FBX9 promoter activity. Results from deletions and truncations defined a minimal promoter for ICK, and revealed that repressors and enhancers differentially regulate ICK versus FBX9 promoter activity. The ICK promoter contains consensus motifs for several FOX-family transcription factors that align when mouse and human are compared using EMBOSS. FOXA1 and FOXA2 increase luciferase activity of a minimal promoter 10-20 fold in HEK293T cells. Consensus sites for TCF7L2 (TCF4) (Gene Id: 6934) are also present in both mouse and human. The expression of beta-catenin increased activity of the minimal promoter approximately 10 fold. ICK reference mRNAs (NM_014920.3, NM_016513) are expressed in low copy number and increased in some breast cancers, using a ten base tag 5'-TCAACCTTAT-3' specific for both ICK transcripts.

Conclusion: ICK and FBX9 are divergently transcribed from a bidirectional promoter that is GC-rich and contains a CpG island. A minimal promoter for ICK contains functional sites for beta-catenin/TCF7L2 and FOXA. These data are consistent with functions that have been proposed for ICK in development and in proliferation or survival of some breast and colon cancers.

Figure 1

Restriction map of genomic DNA between FBX9 and ICK and pGL3 constructs. Indicated fragments were cloned into promoter-less pGL3 for luciferase (Luc) expression. Arrows, start of transcription for reference ICK and FBX9 mRNAs.

Figure 2

FBX9 and ICK are divergently transcribed from a bidirectional promoter. Equal numbers of SKBR3 and AU565 cells were seeded into 96-well plates, transfected with the indicated constructs (Figure 1), then assayed for luciferase activity in each well by the methods described. Data in figures 2 and 3 were obtained by the same procedures. Bar, ± S.D.

Figure 3

The SspI^b to PstI^b segment contains enhancer and suppressor elements. Equal numbers of KM12 and HCT-15 colon cancer cells were seeded into 96-well plates, transfected with the indicated constructs (Figure 1), then assayed for luciferase activity in each well by the methods described. Bar, ± S.D.

Figure 4

The GC-rich EcoRV^b-PstI^b segment is required for ICK reporter activity. Equal numbers of AGS stomach cancer cells and HEK293T cells were seeded into 96-well plates, transfected with the indicated constructs (Figure 1), then assayed for luciferase activity in each well by the methods described. Bar, ± S.D.

Figure 5

Alignment of the human and mouse ICK promoter sequence for the SspI^a to EcoRV^a fragment. Line numbers, distance from ICK start in human or mouse. SspI^a and EcoRV^a sites, double arrows. Underlined, motifs identical in the mouse and human. Gap, omitted bases in alignment. Putative binding sites, inserted name of the factor.

Figure 6

Alignment of the human and mouse ICK promoter sequence for the EcoRV^b-PstI^b fragment. Double arrows, EcoRV^b and PstI^b sites. Single arrow, start sites of indicated reference mRNAs. Putative binding sites, inserted name of the factor.

Figure 7

A minimal promoter for ICK in HEK293TT and HCT-15 cells. Data for ICK were normalized to ICK-9 construct lacking the ICK start site.

Figure 8

Expression of FOXA 1, FOXA2, and β-catenin significantly increase ICK reporter activity. HEK-293T cells in 12-well plates were transiently co-transfected with the ICK-7 luciferase reporter, the control CMV-β-galactosidase reporter, and with expression vectors encoding the various transcription factors or vector control (VC) essentially as described for NFκB reporter assays [64]. The β-galactosidase activities were used to normalize the luciferase values. Co-transfections were also performed with the promoterless ICK-9 luciferase reporter, which served as a negative transcription control. Western blot analyses were performed to ensure that cells expressed the transcription factors (see additional file 1). No suitable antibody was available for CDX1. Data represents the mean + SD of two independent experiments performed in triplicate.