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Mapping and functional characterization of the murine smoothelin-like 1 promoter.

Ulke-Lemée A, Turner SR, Mughal SH, Borman MA, Winkfein RJ, MacDonald JA - BMC Mol. Biol. (2011)

Bottom Line: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle.It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle.Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, University of Calgary, Alberta, Canada.

ABSTRACT

Background: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene.

Results: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence.

Conclusion: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

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EMSAs of the putative MyoD-binding and mirror repeat sequence in the murine Smtnl1 promoter. [γ-32P]-labeled oligonucleotide probes (3 pmol) were incubated with nuclear extracts isolated from murine skeletal muscle (for MyoD, in A) or A7r5 and HEK293 cells (for mirror repeat sequence (MRS), in B). In some experiments, 200-fold excess unlabeled competitors were added prior to incubation with 32P-labeled probes. For supershift analysis, anti-MyoD antibody (1 μg) was introduced with nuclear extracts following the addition of labeled probe. Results are representative of n = 3 independent experiments.
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Figure 5: EMSAs of the putative MyoD-binding and mirror repeat sequence in the murine Smtnl1 promoter. [γ-32P]-labeled oligonucleotide probes (3 pmol) were incubated with nuclear extracts isolated from murine skeletal muscle (for MyoD, in A) or A7r5 and HEK293 cells (for mirror repeat sequence (MRS), in B). In some experiments, 200-fold excess unlabeled competitors were added prior to incubation with 32P-labeled probes. For supershift analysis, anti-MyoD antibody (1 μg) was introduced with nuclear extracts following the addition of labeled probe. Results are representative of n = 3 independent experiments.

Mentions: The highest conservation in the murine Smtnl1 5'-UTR sequence was located between -80 bp and -220 bp relative to the newly derived TSS, suggesting functional importance. Indeed, the region corresponds in part to the strongly activating construct determined with the luciferase reporter assay (i.e., +100 to -118). We searched for transcription factor binding sites (TFBS) in this region using the PATCH program that utilizes the TRANSFAC® database of cis-acting transcription factors [16,17]. Possible mammalian TFBSs in this region include AP2α, SF-1, RXRα, SP1, ER and c-myc amongst others (Figure 4B). Intriguingly, this region of the Smtnl1 promoter also possesses a putative TFBS for MyoD (myogenic differentiation-1), a transcriptional activator of muscle-specific genes [18], located 190 bp upstream of the TSS. MyoD shows highest transcriptional activation when dimerized with another MyoD or other family members (e.g., Sp1, Mef2 and Pbx) [19,20]. Indeed, possible non-canonical sites for MyoD as well as Sp1 are located adjacent to the primary MyoD site in the Smtnl1 promoter. Thus, regulation of Smtnl1 expression via MyoD would explain the robust expression found in skeletal muscle when compared to other tissues. We completed EMSAs with nuclear extracts from mouse skeletal muscle and DNA probes derived from the MyoD site in the Smtnl1 promoter (Figure 5A). Gel shifts were suggestive of protein binding to the Smtnl1 MyoD sequence and could be inhibited with addition of excess unlabelled probe as a competitive inhibitor. Moreover, EMSAs completed in the presence of MyoD antibody exhibited a defined "supershifting" band.


Mapping and functional characterization of the murine smoothelin-like 1 promoter.

Ulke-Lemée A, Turner SR, Mughal SH, Borman MA, Winkfein RJ, MacDonald JA - BMC Mol. Biol. (2011)

EMSAs of the putative MyoD-binding and mirror repeat sequence in the murine Smtnl1 promoter. [γ-32P]-labeled oligonucleotide probes (3 pmol) were incubated with nuclear extracts isolated from murine skeletal muscle (for MyoD, in A) or A7r5 and HEK293 cells (for mirror repeat sequence (MRS), in B). In some experiments, 200-fold excess unlabeled competitors were added prior to incubation with 32P-labeled probes. For supershift analysis, anti-MyoD antibody (1 μg) was introduced with nuclear extracts following the addition of labeled probe. Results are representative of n = 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050715&req=5

Figure 5: EMSAs of the putative MyoD-binding and mirror repeat sequence in the murine Smtnl1 promoter. [γ-32P]-labeled oligonucleotide probes (3 pmol) were incubated with nuclear extracts isolated from murine skeletal muscle (for MyoD, in A) or A7r5 and HEK293 cells (for mirror repeat sequence (MRS), in B). In some experiments, 200-fold excess unlabeled competitors were added prior to incubation with 32P-labeled probes. For supershift analysis, anti-MyoD antibody (1 μg) was introduced with nuclear extracts following the addition of labeled probe. Results are representative of n = 3 independent experiments.
Mentions: The highest conservation in the murine Smtnl1 5'-UTR sequence was located between -80 bp and -220 bp relative to the newly derived TSS, suggesting functional importance. Indeed, the region corresponds in part to the strongly activating construct determined with the luciferase reporter assay (i.e., +100 to -118). We searched for transcription factor binding sites (TFBS) in this region using the PATCH program that utilizes the TRANSFAC® database of cis-acting transcription factors [16,17]. Possible mammalian TFBSs in this region include AP2α, SF-1, RXRα, SP1, ER and c-myc amongst others (Figure 4B). Intriguingly, this region of the Smtnl1 promoter also possesses a putative TFBS for MyoD (myogenic differentiation-1), a transcriptional activator of muscle-specific genes [18], located 190 bp upstream of the TSS. MyoD shows highest transcriptional activation when dimerized with another MyoD or other family members (e.g., Sp1, Mef2 and Pbx) [19,20]. Indeed, possible non-canonical sites for MyoD as well as Sp1 are located adjacent to the primary MyoD site in the Smtnl1 promoter. Thus, regulation of Smtnl1 expression via MyoD would explain the robust expression found in skeletal muscle when compared to other tissues. We completed EMSAs with nuclear extracts from mouse skeletal muscle and DNA probes derived from the MyoD site in the Smtnl1 promoter (Figure 5A). Gel shifts were suggestive of protein binding to the Smtnl1 MyoD sequence and could be inhibited with addition of excess unlabelled probe as a competitive inhibitor. Moreover, EMSAs completed in the presence of MyoD antibody exhibited a defined "supershifting" band.

Bottom Line: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle.It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle.Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, University of Calgary, Alberta, Canada.

ABSTRACT

Background: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene.

Results: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence.

Conclusion: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

Show MeSH