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. 2009 May 5:10:37.
doi: 10.1186/1471-2199-10-37.

Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing

Atif M Nakhla  1 , Daniel J Hryb, William Rosner, Nicholas A Romas, Zhaoying Xiang, Scott M Kahn
Affiliations

Human sex hormone-binding globulin gene expression- multiple promoters and complex alternative splicing

Atif M Nakhla et al. BMC Mol Biol. .

Abstract

Background: Human sex hormone-binding globulin (SHBG) regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (P(L)). The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (P(T)), and two minor transcripts.

Results: We report that transcriptional expression of the human SHBG gene is far more complex than previously described. P(L) and P(T) direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons 4, 5, 6, and/or 7. We mapped two transcriptional start sites downstream of P(L) and P(T), and present evidence for a third SHBG gene promoter (P(N)) within the neighboring FXR2 gene; PN regulates the expression of at least seven independent SHBG gene transcripts, each possessing a novel, 164-nt first exon (1N). Transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the LNCaP, MCF-7, and HepG2 cell lines. Each expresses the SHBG transcript, albeit in varying abundance. Alternative splicing was more pronounced in the cancer cell lines. P(L)- P(T)- and P(N)-derived transcripts were most abundant in liver, testis, and prostate, respectively. Initial findings reveal the existence of a smaller immunoreactive SHBG species in LNCaP, MCF-7, and HepG2 cells.

Conclusion: These results extend our understanding of human SHBG gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant SHBG gene expression may play in cancer.

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Figures

Figure 1
Figure 1
Schematic diagram of the contiguous human SHBG/ABP transcript and alternatively spliced transcripts arising from the downstream promoter, PL. A structure/function diagram for the eight exon long SHBG/ABP transcript is shown above. The nucleotide positions of each exon are at the top (the exon 1 start codon defined as beginning with nucleotide +1); below that, the number of amino acids encoded by each exon. Conserved exons are numbered in white, alternatively spliced exons in yellow. Exon boundaries are denoted by horizontal white rectangles. Shown in red is the region of the SHBG/ABP transcript encoding the 29 amino acid long signal peptide, in light blue, the 383 amino acid long SHBG/ABP monomer. Steroid binding determinants in the mature SHBG protein are denoted by black rectangles, those forming the dimerization domain are blue, and that encoding the decapeptide sequence known to mediate binding of SHBG to RSHBG is purple. Below, in black, are linear depictions of the two previously described human SHBG gene transcripts originating from PL; the four novel transcripts described in this study are depicted in grey. Transcripts are identified on the left by missing exons. Three of five alternatively spliced PL transcripts retain the SHBG/ABP reading frame, whereas those lacking exon 7 alone and exons 4–7 undergo identical frameshifts within exon 8 that may affect their inherent stabilities (see discussion).
Figure 2
Figure 2
Short range RT-PCR assays for human SHBG gene expression. A. SHBG exon 5–8 assay. First strand cDNAs were generated from MCF-7, MCF-7-Tr LNCaP, HepG2 cells and normal human testis tissue using an oligo dT primer (see methods). Three RT-PCR transcripts were generated in the MCF-7 and LNCaP lanes, and four in the HepG2 and testis lanes. MCF-7-Tr is an MCF-7 clone stably transfected with a plasmid constitutively expressing large amounts of SHBGL. All bands were reamplified and sequenced. SHBG RT-PCR transcript structures are given on the right. Note- this is a merged figure- the DNA marker and transfection control lanes were consolidated from a different part of the gel. B. SHBG exon 2–8 assay. PCR primers, specific for exon 2 and exon 8, were used to amplify cDNAs prepared from HepG2, LNCaP, MCF-7, MCF-7-Tr, and normal human testis, liver, prostate, breast, and brain tissues. All bands from HepG2, LNCaP, MCF-7, and testis were reamplified and sequenced. Bands labeled "*" produced sequences that were inconsistent with the sizes of the transcripts, and which were identical to other characterized sequences. DNA marker sizes, in base pairs, are given on the left. RT-PCR fragment transcript structures and sizes are given on the right. Note- this is a merged figure- the transfection control lane was taken from a different part of the gel.
Figure 3
Figure 3
Summary of RT-PCR results. Semiquantitative representation of the relative amounts of each RT-PCR fragment. For ease of comparison, the abundance of each RT-PCR fragment in the exon 2–8 and the exon 1–8 RT-PCR assays has been assigned a value, denoted by the following- "+++++" most abundant, down to "+" detectable, "+/-" barely detectable, and "-" not detectable. For each individual assay, transcript structures were determined by sequence analysis of designated RT-PCR fragments (see Results). These were used to assign structures to similar sized RT-PCR fragments from remaining samples. *: This band was a mixture of 1N-8-exon 4 sequences and those of an ambiguous sequence. "?"-inconsistent appearance of fragment, presumably due to low abundance.
Figure 4
Figure 4
Configuration of A) human SHBG exon 1L and B) exon 1T. Transcriptional start sites, as determined in this study, are in bold letters and are denoted by a "≥ ". For both exons, position +1 is assigned to the distal, upstream start site. The exon 1L proximal start site is at position +42, the exon 1T proximal start site is at position +23. Upstream sequences are in italics. Previously reported human transcriptional start sites are underlined; not shown are two additional exon 1L start sites mapped at positions -114 and -125 [22]. Transcriptional start sites mapped in transgenic mice are in lower case letters [23]. "r" and "b" denote the corresponding positions of the major exon 1L rat [43] and rabbit [44] transcriptional start sites, respectively.
Figure 5
Figure 5
A- Sequence of human SHBG exon 1N and species alignment. Shown is an alignment between the full human SHBG exon 1N sequence and homologous sequences from mouse, rat, rabbit and dog obtained from Genbank. The putative human ATG start codon is in bold and underlined. The TGA stop codon is in bold and italics. Conserved nucleotides between species are capitalized, differences are in lower case letters. The human exon 1N open reading frame is predicted to be only 3 amino acids long. B- Sequence of rat SHBG exon 1A and species alignment. Shown is an alignment between the rat SHBG exon 1A sequence and homologous human and mouse sequences obtained from Genbank. The putative rat ATG start codon is in bold and underlined. Conserved nucleotides between species are capitalized, differences are in lower case letters.
Figure 6
Figure 6
RT-PCR analysis of transcripts including exon 1L (top), exon 1T (middle), and exon 1N (bottom). Separate individual RT-PCR amplifications were performed using 5' primers specific for exon 1L, 1T, and 1N, and a common exon 8 3' primer. Products were electrophoresed through a 1% agarose gel. All fragments were reamplified and sequenced. Bands labeled "*" produced ambiguous sequences that were inconsistent with the sizes of the transcripts, and which were identical to other characterized sequences (the smallest "*" fragment in the 1T-8 assay gave an unrelated sequence). DNA marker sizes, in base pairs, are given on the left. RT-PCR fragment transcript structures and sizes are given on the right. Note- 5A is a merged figure from two experiments to more clearly show the prostate 1L-8 RT-PCR fragment. Certain HepG2, MCF-7, testis and prostate fragments showed varied appearance in the 1N-8 assay, presumably due to their low relative abundance (Figure 3).
Figure 7
Figure 7
Relative SHBG Gene Expression (Total and Promoter-specific) Among Human Cancer Cell Lines and Normal Tissues. Quantitative RT-PCR (qPCR) was performed on total cellular RNA samples isolated from the indicated cell lines (in triplicate) and from normal tissues (in duplicate), using primers specific for SHBG exons 1L and 2, 1T and 2, 1N and 2, or exons 2 and 3 as described in the Experimental Procedures. Each sample was analyzed by qPCR in triplicate (nine total measurements for each cell line per assay, six for each tissue). Ct values represent the number of cycles required to reach an arbitrary point on the exponential part of the qPCR curves. "Normalized Ct" values for each of the 1L-2, 1T-2, 1N-2 and 2–3 assays represent mean Ct values for a given sample, normalized to the mean GAPDH Ct value for that same sample. SD: Standard deviation, calculated from the averages of the total measurements. Relative transcript abundance is presented as the ratio of Ct means; an arbitrary value of 1 is assigned to MCF-7.
Figure 8
Figure 8
Western blot analysis of SHBG protein expression in HepG2, LNCaP, and MCF-7 cells. Protein extracts (20 μg each) prepared from HepG2, LNCaP, and MCF-7 cells, and 10 picograms of purified SHBG protein isolated from human plasma using a steroid affinity column, were electrophoresed through 10% LongLife polyacrylamide gels (Gradipore-VWR), transferred to PVDF membranes, and hybridized to either a rabbit anti-human SHBG polyclonal antibody (top, WAK-S1012-53, WAK-Chemie, Germany) or a rabbit anti-actin affinity purified polyclonal antibody (bottom, A2066, Sigma). Molecular weight (in kilodaltons) marker positions are given on the left. The top band in HepG2 and LNCaP cells migrates with a molecular weight of approximately 44–46 kD, while the bottom band migrates with a molecular weight of approximately 33–35 kD. The two larger bands in MCF-7 migrate with molecular weights of 51–53 kD and 48–50 kD, respectively. Note- the size marker appears to have run a bit slowly on the top gel, as the purified SHBG is slightly smaller than prior reports of 50–52 kD.
Figure 9
Figure 9
The human SHBG gene locus on chromosome 17p13.1 and its transcriptional expression. Top: Exon structures of the SHBG gene and its neighboring genes are given as vertical lines. Transcriptional polarity is denoted by arrows. The polarity (designated by arrows) of the SHBG gene is opposite that of the SAT2 and FXR2 genes. SHBG upstream exon 1T abuts the SAT2 gene, and exon 1N is located within intron 1 of the FXR2 gene. Note the close proximity of the p53 gene. Bottom: An expanded structure of the human SHBG gene, showing the three SHBG gene promoters, PL, PT, and PN, the three first exons, 1L, 1T, and 1N, and downstream exons 2–8. Numbers refer to relative positions along chromosome 17. Below is a representation of the structures of the 19 known human SHBG gene transcripts (boxes represent exons). Transcript structures are given on the right. Those representing the two major transcripts (SHBG/ABP and SHBG-T) and two minor transcripts (1L-8 lacking exon 7, and 1T-8) from prior reports are underlined. Alternative splicing of exon 4 from 1L-8 and 1T-8 transcripts was not specifically detected; these putative transcripts are presented in parentheses.
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References

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