Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts

Abstract

The effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. We have identified upstream exons of the human (h) VDR gene that are incorporated into variant transcripts, two of which encode N-terminal variant receptor proteins. Expression of the hVDR gene, which spans more than 60 kb and consists of at least 14 exons, is directed by two distinct promoters. A tissue-specific distal promoter generates unique transcripts in tissues involved in calcium regulation by 1, 25-(OH)2D3 and can direct the expression of a luciferase reporter gene in a cell line-specific manner. These major N-terminal differences in hVDR transcripts, potentially resulting in structural differences in the expressed receptor, may contribute to cellular responsiveness to 1,25-(OH)2D3 through tissue differences in the regulation of VDR expression.

Figure 1

(A) Human VDR gene locus. Four overlapping cosmid clones were isolated from a human lymphocyte genomic library (Stratagene) and directly sequenced. Clone J5 extends from the 5′ flanking region to intron 2; AE, from intron 1b to intron 5; D2, from intron 3 to the 3′ UTR; WE, from intron 6 through the 3′ flanking region. Sequence upstream of exon 1f was obtained by anchored PCR from genomic DNA. (B) Structure of hVDR transcripts. Transcripts 1–5 originate from exon 1a. Transcript 1 corresponds to the published cDNA (5). Transcripts 6–10 originate from exon 1d and transcripts 11–14 originate from exon 1f. Boxed numbers indicate the major transcript (based on the relative intensities of the multiple PCR products) within each exon-specific group of transcripts generated with a single primer set. While all transcripts have a translation initiation codon in exon 2, exon 1d transcripts have the potential to initiate translation upstream in exon 1d, with transcripts 6 and 9 encoding VDR proteins with extended N termini. (C) N-terminal variant proteins encoded by novel hVDR transcripts. Transcript 1 corresponds to the published cDNA sequence (5) and encodes the 427-aa hVDR protein. Transcripts 6 and 9 code for a protein with an extra 50 aa or 23 aa, respectively, at the N-terminal. The 23 aa of the hVDR A/B domain are shown in bold.

Figure 2

RT-PCR analysis of expression of variant hVDR transcripts. (A) Exon 1a transcripts (220 bp, 301 bp, 342 bp, 372 bp, and 423 bp). (B) Exon 1d transcripts (224 bp, 305 bp, 346 bp, 376 bp, and 427 bp). (C) Exon 1f transcripts (228 bp, 309 bp, 387 bp, and 468 bp). RT-PCR was carried out with exon 1a-, 1d-, or 1f-specific forward primers and a common reverse primer in exon 3. The sizes of the PCR products and the pattern of bands are similar in A and B by virtue of the identical splicing pattern of exon 1a and 1d transcripts and the fact that primers were designed to generate PCR products of comparable sizes. All tissues and cell lines are human in origin.

Figure 3

Functional analysis of sequence-flanking exons 1a and 1d (A) and exon 1f (B) in NIH 3T3 (solid bars) and COS 7 cells (open bars). The parent vector pGL3basic was used as a promoterless control, and a promoter-chloramphenicol acetyltransferase (CAT) gene reporter construct was cotransfected as an internal control for transfection efficiency in each case. The activity of each construct was corrected for transfection efficiency and for the activity of the pGL3basic empty vector control and expressed as a percentage of the activity of the construct 1a(−488,+75) ± SEM of at least three separate transfections. Exon 1a and 1d flanking constructs are defined in relation to the transcription start site of exon 1a, designated +1, which lies 54 nt upstream of the published cDNA (5). Exon 1f flanking constructs are defined relative to the exon 1f transcription start site, designated +1. Transcription start sites were determined by the 5′ termini of the longest RACE clones. The open box corresponds to the GC-rich region.