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

Sturgill TW, Stoddard PB, Cohn SM, Mayo MW - Mol. Cancer (2010)

Bottom Line: The ICK promoter contains consensus motifs for several FOX-family transcription factors that align when mouse and human are compared using EMBOSS.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pharmacology and Internal Medicine, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908, USA. tws7w@virginia.edu

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.

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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.
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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.

Mentions: To enable initial studies of transcription factors, we chose a minimal ICK promoter for use in HEK293T cells. Activity in HEK293T (Fig. 3) and HCT-15 (Fig. 4) cells did not depend greatly on SspIa-SspIb and SspIb-EcoRVa fragments. To compare data from these lines, we normalized our promoter data for ICK constructs to ICK-9 (Fig. 7). Activity of the full ICK promoter (ICK-1) is increased 13-14 fold in both of these lines. The normalized results for truncations from the 5' end show that elements required for luciferase activity in HEK293T and HCT-15 cells reside in the EcoRVa-EcoRVb (611 nt) fragment and the EcoRVb-Pst1 (503 nt) fragment. ICK-6 and ICK-7 also retain the majority of reporter activity for ICK in the other cell lines. The first and second EcoRV cut sites are 1195 and 587 nt, respectively, from the predicted transcription start site of human ICK. Two alternative reference mRNAs (NM_016513, NM_014920) use the same start site GGAAAAC within PstIb-ApaIc. We chose the smaller construct ICK-7 (~0.8 kb), with ~0.6 kb of 5' sequence, as the minimal promoter to study in the next experiments.


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

Sturgill TW, Stoddard PB, Cohn SM, Mayo MW - Mol. Cancer (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2876993&req=5

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.
Mentions: To enable initial studies of transcription factors, we chose a minimal ICK promoter for use in HEK293T cells. Activity in HEK293T (Fig. 3) and HCT-15 (Fig. 4) cells did not depend greatly on SspIa-SspIb and SspIb-EcoRVa fragments. To compare data from these lines, we normalized our promoter data for ICK constructs to ICK-9 (Fig. 7). Activity of the full ICK promoter (ICK-1) is increased 13-14 fold in both of these lines. The normalized results for truncations from the 5' end show that elements required for luciferase activity in HEK293T and HCT-15 cells reside in the EcoRVa-EcoRVb (611 nt) fragment and the EcoRVb-Pst1 (503 nt) fragment. ICK-6 and ICK-7 also retain the majority of reporter activity for ICK in the other cell lines. The first and second EcoRV cut sites are 1195 and 587 nt, respectively, from the predicted transcription start site of human ICK. Two alternative reference mRNAs (NM_016513, NM_014920) use the same start site GGAAAAC within PstIb-ApaIc. We chose the smaller construct ICK-7 (~0.8 kb), with ~0.6 kb of 5' sequence, as the minimal promoter to study in the next experiments.

Bottom Line: The ICK promoter contains consensus motifs for several FOX-family transcription factors that align when mouse and human are compared using EMBOSS.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Pharmacology and Internal Medicine, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908, USA. tws7w@virginia.edu

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.

Show MeSH
Related in: MedlinePlus