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. 2006 Nov 13;580(26):6217-23.
doi: 10.1016/j.febslet.2006.10.025. Epub 2006 Oct 19.

Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase

Stephen C Linn  1 , Lindsay M Andras, Hee-Sook Kim, Jia Wei, M Marek Nagiec, Robert C Dickson, Alfred H Merrill Jr
Affiliations

Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase

Stephen C Linn et al. FEBS Lett. .

Abstract

A series of luciferase reporter constructs was prepared from a 1035-bp fragment of mouse genomic DNA flanking the 5'-coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full-length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site-directed mutagenesis indicated that the proximal 335 bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to stimulate transcription in a cooperative manner, and several additional elements whose activity cannot be accounted for by known factor binding sites. These findings provide insight into the control mechanisms for transcription of mammalian SPTLC2.

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Figures

Fig. 1
Fig. 1
Nucleotide sequence of the mouse SPTLC2 promoter region. Murine genomic sequences are aligned with homologous sequences from human chromosome 14 (GenBank accession number AF111168). Gaps have been introduced where necessary to maintain alignment and identical sequences are shaded. Nucleotides are numbered relative to the presumptive transcription initiation site in the mouse gene (see text), and a sequence matching the initiator (Inr) element consensus PyPyA+1N(T/A)PyPy is labeled. A downstream sequence that matches the downstream promoter element (DPE) consensus at 4 of 5 positions is shown in parentheses. Labeled bars indicate the positions of potential transcription factor binding sites. Sites containing deviations from conserved nucleotides in the consensus sequence are shown in parentheses; potential binding sites with differences in nucleotides considered to be invariant, or nearly so, were ignored.
Fig. 2
Fig. 2
Sequence elements required for basal transcription are located within the first 335 bp of the mouse SPTLC2 promoter region. A: Luciferase reporter constructs containing promoter segments extending from +80 to the indicated 5′ endpoints were transfected into NIH3T3 cells and luciferase activity was assayed after 24 h. Data were normalized to the reporter activity of a cotransfected pRL-TK plasmid and background luciferase activity determined by transfection of a promoterless vector was subtracted. Activities are expressed relative to the −955 bp promoter and results shown represent the means ± S.E.M. of at least three independent experiments. Decreases in promoter activity that are significant relative to the prior construct in the deletion series (single-factor Anova) are indicated by the appropriate P value. B: Promoter deletion mutants terminating between positions −230 and −357 were assayed as described above. Locations of potential transcription factor binding sites in relationship to the deletion endpoints are indicated. Activities are expressed relative to the −357 bp promoter and results shown represent the means ± S.E.M. of at least nine independent experiments. Decreases in promoter activity that are significant relative to the prior construct in the deletion series (single-factor Anova) are indicated by the appropriate P value. Promoter activity of the −317 bp mutant was significantly different from the −357 bp control (P < 0.05) as well as from the −335 bp mutant.
Fig. 3
Fig. 3
Two consensus GC boxes at positions −51 and −109 account for basal SPTLC2 promoter activity in the −34 to −127 bp region. A: NIH3T3 cells were transfected with luciferase reporter constructs containing the wild type −955 bp promoter or promoters containing mutations in the −109 bp, −51 bp, or both GC-boxes. Control cells were transfected with a −127 bp deletion mutant lacking the upstream promoter domain and a −34 bp mutant in which both GC-boxes are removed. Luciferase activity was assayed after 24 h and normalized to the reporter activity of a cotransfected pRL-TK plasmid. Background luciferase activity determined by transfection of a promoterless vector was subtracted and all activities are expressed relative to the −955 bp wild type (wt) promoter. Results shown represent the means ± S.E.M. of three independent transfections. B: Cells were transfected with a −127 bp promoter deletion mutant containing the wild type sequence or the GC-box mutations described above. Control cells were transfected with the −357 bp or −34 bp promoter construct. Luciferase activity was assayed after 24 h and normalized to the reporter activity of a cotransfected pRL-TK plasmid. Background luciferase activity determined by transfection of a promoterless vector was subtracted and activities are expressed relative to the wild type −955 bp promoter. Results shown represent the means ± S.E.M. of three independent transfections.
Fig. 4
Fig. 4
Mutation of CCAAT sites at −332 and −248 increases SPTLC2 promoter activity. NIH3T3 cells were transfected with luciferase reporter constructs containing the wild type (wt) −955 bp promoter or substitution mutations in the −248 bp sense strand or −332 bp antisense strand CCAAT sequences. Luciferase reporter activity was assayed after 24 h and normalized to the activity of a cotransfected pRL-TK plasmid. Background luciferase activity determined by transfection of a promoterless vector was subtracted from all values. Activities are expressed relative to the wild type −955 bp promoter and results shown represent the means ± S.E.M. of three independent transfections. Reporter gene expression from both mutants was significantly greater than from the wild type control (P << 0.001 in single-factor Anova) but the mutants were not significantly different from one another (P > 0.10).
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