The Samd9L gene: transcriptional regulation and tissue-specific expression in mouse development

Abstract

Normophosphatemic familial tumoral calcinosis (NFTC) is caused by mutations in the SAMD9 gene. This gene is absent in mouse, while there is a murine paralog, Samd9-like (Samd9L). To clarify the relationships between SAMD9 and SAMD9L, we investigated the transcriptional regulation and expression pattern of mouse Samd9L. An ∼1.5-kb mouse Samd9L promoter fragment was cloned, and a series of 5' deletion constructs were linked to a luciferase reporter gene. All constructs showed significant activity in transfected epithelial cells and mouse fibroblasts, and the presence of regulatory cis-elements as close as 87 bp upstream of the transcription start site was identified. Ras-responsive element binding protein 1 (Rreb-1) was identified in this region by protein-DNA binding array. The expression of Samd9L was upregulated by calcitonin, and this was preceded by a significant increase in the expression of Rreb-1 mRNA. Quantitative real-time PCR analysis of Samd9L revealed near-ubiquitous expression, with the highest level in the kidney. Tissue-specific expression was also confirmed both by in situ β-gal staining and quantitative enzymatic activity assay in a transgenic Samd9L(+/-) mouse in which the LacZ gene replaced exon 2 in the Samd9L gene. These findings assist in understanding the regulation of Samd9L in the context of its paralogous gene, SAMD9, which harbors mutations in NFTC.

Figure 1. Samd9L expression is tissue-specific and developmentally regulated

(a) Samd9L mRNA expression was assessed by PCR using a pre-standardized cDNA library of multiple mouse tissues. The represented tissues are as follows: 1. Brain; 2. Heart; 3. Kidney; 4. Spleen; 5. Thymus; 6. Liver; 7. Stomach; 8. Small Intestine; 9. Muscle; 10. Lung; 11. Testis; 12. Skin; 13. Adrenal Gland; 14. Pancreas; 15. Uterus; 16. Prostate Gland; 17. Embryo/8.5 day; 18. Embryo/9.5 day; 19. Embryo/12.5 day; 20. Embryo/19 day. (b) The kidneys were harvested from the mice at the different ages. RT-PCR analysis revealed that the expression of mouse Samd9L in the kidneys increased with age when the mice at the age of 0 (newborn), 2, 4 and 40 weeks were examined. (c) The levels of Samd9L mRNA expression were quantified by determining the pixel intensities of the bands of RT-PCR products on the images in (b) using a computer program (ImageQuant, Molecular Dynamics, Sunnyvale, CA), and normalized with those of the corresponding internal actin controls. Data from three independent measurements were used (mean ± SD). The relative expression of mouse Samd9L was calculated using the mRNA level of mouse Samd9L in the newborns as 1 (*p< 0.05).

Figure 2. Serially truncated 5’ deletion constructs of the mouse Samd9L promoter display significant activities in various cell lines

(a) Serial deletion constructs of the mouse Samd9L 5’- flanking region were inserted upstream of firefly luciferase gene (Luc) in the reporter plasmid pGL3-Basic. The numbers on the left indicate the 5’ position of the construct, starting from the transcription start site (+1; arrow). (b) The Samd9L promoter constructs (0.75 µg) described in (a) or the control pGL3-Basic vector were cotransfected with 0.25 µg of pRSV-β-galactosidase reporter plasmid into the indicated cell types using FuGENE6 reagent (Roche). The cells were lysed 50 hours later and assayed for luciferase and β-galactosidase activities. Luciferase activity was divided by β-galactosidase activity to correct for transfection efficiency. The data are presented as the mean ± SD of three separate experiments each performed in triplicate. TCMK-1, mouse kidney epithelial cell line; MLE-10, mouse liver epithelial cell line; HEK293, human embryonic kidney epithelial cell line; NIH3T3, mouse skin fibroblast cell line.

Figure 3. Protein/DNA array identifies select transcription factors binding to the mouse Samd9L promoter region

(a) Hybridization signals of the transcription factors bound to the p-87 promoter fragment identified in nuclear extracts of NIH3T3 cells. The TranSignal protein/DNA filter contains 96 transcription factors, each tested in duplicate (adjacent horizontal dots). (b) The transcription factors identified by the protein/DNA array as binding to Samd9L promoter region are indicated on shaded background. The selected transcription factors whose putative recognition sites that were identified by TFSEARCH (version 1.3, Kyoto University, Japan) in the p-87 promoter fragment are outlined by black frame in (a and b). Insufficient signal was noted for those factors shown on the white background. (c) The sequence extending from −87 to +98 was scanned for transcription factor-binding sites by using TFSEARCH. The putative recognition sites for select transcription factors identified in DNA are indicated below the sequence. The transcription initiation site is referred to by +1.

Figure 4. Calcitonin enhances the expression of mouse Samd9L and Rreb genes in NIH 3T3 cells in vitro

RT-PCR was performed using total RNA isolated from NIH3T3 cells cultured with 200 ng/L calcitonin for the indicated time periods using primers specific for Samd9L, Rreb and Gapdh, as described in Materials and Methods. The data were normalized to the Gapdh endogenous control and are presented as fold change as compared to untreated cells (0 time point). Samd9L expression is presented in the top panel (a) and Rreb expression in the bottom panel (b) Asterisks indicate statistical significance (p < 0.05) when compared to the corresponding controls.

Figure 5. Mutations in the Rreb binding site sequences alter the activity of Samd9L gene promoter

(a) Sequences for the published (Thiagalingam et al, 1996) consensus sequence for the RREB binding site and WT sense strand of the p-87 plasmid construct (sequence homology boxed), as well as mutated plasmids (Mut 1, Mut 2, Mut 3) developed by site-directed mutagenesis with mutant primers, as described in Materials and Methods, are indicated in the figure. (b) The WT and mutant constructs were cotransfected with pRSV-β-galactosidase plasmid (0.75 µg to 0.25 µg, respectively) into NIH 3T3 cells using FuGENE6 (Roche), lysed after 50 hours of incubation, and then assayed for luciferase activity (Luc) which was normalized by β-galactosidase activity to correct for transfection efficiency. The data are presented as the mean ± SD of three independent experiments each performed in triplicate. Asterisks indicate statistical significance (p < 0.05) when compared to the WT construct.

Figure 6. Assessment of Samd9L reveals tissue-specific expression in vivo

(a) Various organs were harvested from Samd9L^+/− mice, in which the LacZ gene replaced exon 2 in the Samd9L gene in one allele, and were analyzed by in situ β-Gal staining. (b) Tissue-specific expression was analyzed by quantitative enzymatic activity assay in a transgenic Samd9L^+/− mouse. The β-galactosidase activity in each tissue of the wild-type mice was used as the baseline, and the data were expressed after normalization to the corresponding baselines and to the protein content of each extract. The highest β-galactosidase enzymatic activity was observed in the kidney and spleen. (c) Paraffin sections of the β-Gal stained kidney confirmed localization in both proximal and distal tubules. (d) Paraffin sections of WT mouse kidney was stained with Samd9L primary antibody and Texas Red secondary antibody and counter-stained with DAPI (right panel), or stained only with Texas Red secondary antibody and DAPI (left panel). The Samd9L was expressed in the tubules (arrows) but not in the glomerulis (in rectangles). Scale bar, 100 µm.