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. 2003 Oct 28;100(22):12841-6.
doi: 10.1073/pnas.2134464100. Epub 2003 Oct 8.

An endogenous retroviral long terminal repeat is the dominant promoter for human beta1,3-galactosyltransferase 5 in the colon

Catherine A Dunn  1 , Patrik Medstrand, Dixie L Mager
Affiliations

An endogenous retroviral long terminal repeat is the dominant promoter for human beta1,3-galactosyltransferase 5 in the colon

Catherine A Dunn et al. Proc Natl Acad Sci U S A. .

Abstract

LTRs of endogenous retroviruses are known to affect expression of several human genes, typically as a relatively minor alternative promoter. Here, we report that an endogenous retrovirus LTR acts as one of at least two alternative promoters for the human beta1,3-galactosyltransferase 5 gene, involved in type 1 Lewis antigen synthesis, and show that the LTR promoter is most active in the gastrointestinal tract and mammary gland. Indeed, the LTR is the dominant promoter in the colon, indicating that this ancient retroviral element has a major impact on gene expression. Using colorectal cancer cell lines and electrophoretic mobility-shift assays, we found that hepatocyte nuclear factor 1 (HNF-1) binds a site within the retroviral promoter and that expression of HNF-1 and interaction with its binding site correlated with promoter activation. We conclude that HNF-1 is at least partially responsible for the tissue-specific activation of the LTR promoter of human beta 1,3-galactosyltransferase 5. We demonstrate that this tissue-specific transcription factor is implicated in the activation of an LTR gene promoter.

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Figures

Fig. 1.
Fig. 1.
Exon 1 of β3Gal-T5 is derived from an endogenous retroviral LTR. Exon (E) sequences are shown in uppercase reverse type, with all other sequences in lowercase. The translation initiation codon at the beginning of exon 4 and the 5-bp direct repeats flanking the LTR are shown in italics. The ERV-L LTR sequence is shown in bold and framed with a solid black line. Dotted white lines frame the portion of exon 1 included in splicing variant 1, but not variant 2; three minor splicing variants, which contain additional exon sequences, are not shown (see ref. 15). Gray boxes, putative HNF-1 binding sites; underlined text, putative CCAAT box; dashed underlined text, putative Jun binding site; unfilled boxes, putative Sp1 binding sites.
Fig. 2.
Fig. 2.
Detection of β3Gal-T5 transcripts initiating within the LTR and native promoters. (A) Total cDNAs derived from a range of normal human tissues were used in RT-PCR assays, with primers specific for GAPDH, the β3Gal-T5 ORF, and β3Gal-T5 transcripts initiating within the LTR or native promoter. Approximate molecular weights are indicated on the left. (B) Southern blot of the gel shown in the third panel in A. The blot was probed with a labeled oligonucleotide specific for β3Gal-T5 exon 4.
Fig. 3.
Fig. 3.
Genomic structure and nucleotide sequence of transcripts initiating from the native promoter of β3Gal-T5. (A) Structure of the β3Gal-T5 locus. Exons are boxed and numbered. The ERV-L LTR is represented by an arrow. β3Gal-T5 transcripts initiating from the native promoter (NP) and LTR promoter (LP-splicing variant 1 only) are shown schematically below. The diagram is not to scale. (B) Nucleotide sequence of transcripts initiating from the β3Gal-T5 native promoter. Exon sequences are shown in uppercase reverse type, with all other sequences in lowercase. The translation initiation codon is shown in italics.
Fig. 4.
Fig. 4.
Contribution of the LTR promoter to β3Gal-T5 expression in human tissues. Primers were used in real-time PCR assays to amplify transcripts specific for GAPDH, total β3Gal-T5, and β3Gal-T5 transcripts initiating within the LTR promoter from cDNAs derived from normal human tissues. Gray bars represent the relative abundance of total β3Gal-T5 transcripts normalized to GAPDH levels ± SEM. Black bars depict the contribution of the LTR promoter to total β3Gal-T5 transcription ± SEM. In some cases, the error bars are too small to see. Relative levels of transcripts are indicated above the bars where necessary. Assays were carried out in duplicate and repeated twice.
Fig. 5.
Fig. 5.
5′ deletion analysis of the LTR promoter in human colorectal cancer cell lines. ERV-L LTR nucleotides 1–522 and five sequentially 5′ deleted LTRs were cloned into the pGL3B luciferase reporter vector. Plasmids were transiently transfected into DLD-1 and LoVo cells. Luciferase activities are shown relative to the internal control and then to pGL3B to demonstrate fold activation. Bars represent the mean of four independent transfections ± SEM.
Fig. 6.
Fig. 6.
Site-directed mutagenesis of putative HNF-1 binding sites within the LTR. (A) Mutation of two putative HNF-1 binding sites within the LTR 1–522 reporter construct. Plasmids were transiently transfected into DLD-1 and LoVo cells. Luciferase activities are shown as fold activation as before. Bars represent the mean of four independent transfections ± SEM. H, putative HNF-1 binding site. White type represents mutation of the putative binding site. (B) The sequence of LTR nucleotides 7–47 in each construct is indicated, with mutated nucleotides in uppercase bold type.
Fig. 7.
Fig. 7.
HNF-1 binds the LTR promoter specifically in the LoVo cell line. A radiolabeled oligonucleotide containing putative HNF-1 binding site 2 was incubated with nuclear extracts containing 5 μg of protein from DLD-1 (D) or LoVo (L) cells. Extracts were preincubated with a 100-fold molar excess of WT or mutated competitor oligonucleotides, or with an antibody reactive with HNF-1, as indicated.
Fig. 8.
Fig. 8.
Detection of HNF-1α transcripts in human tissues and colorectal cancer cell lines. Total cDNAs derived from normal human tissues and the DLD-1 and LoVo cell lines were used in RT-PCR, with primers specific for GAPDH (Upper) or HNF-1α (Lower). Approximate molecular weights are indicated on the left.
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