Mapping and functional characterization of the murine Smoothelin-like 1 promoter

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

Assembly of retinal rod or cone Na+/Ca2+/K+-exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs

Proper control of intracellular free Ca(2+) is thought to involve subsets of proteins that co-localize to mediate coordinated Ca(2+) entry and Ca(2+) extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca(2+) influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na(+)/Ca(2+)-K(+) exchanger (NCKX) is the only Ca(2+) extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments

Expression of DCT following morpholino knockdown of SLC24A5.

<p>(A) Non injected side. (B) 100ng morpholino injected side. (C) Embryo in (A) following DCT in situ. (D) Embryo in (B) following DCT in situ. (E,F) Non injected controls following DCT in situ, n = 19.</p

Rescuing with Xenopus mutants.

<p>Embryos were injected with 100ng morpholino and 5ng cRNA of the mutant constructs. Embryos were allowed to develop to stage 38. The experiments were repeated in triplicate and the results were combined, n values shown at top of graph.</p

Rescuing the morpholino with chimeric constructs.

<p>5ng RNA of the constructs was co-injected with 100ng morpholino, the embryos were raised to stage 38. The experiments were repeated in triplicate and the results were combined, n values shown at top of graph. (A-D) shows schematics of chimera rescue contructs. (E) Embryos were scored according to the scale in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180465#pone.0180465.g002" target="_blank">Fig 2</a>. Kruskal Wallis revealed a significant difference between 100ng ATG MO alone and the NCKX5 and NCKX5 ntl2 constructs, p = 0.01 and p = 0.022 respectively.</p

Expression of SLC24A5 in <i>X</i>.<i>laevis</i>.

<p>(A-K) Whole mount in situ hybridisation, (L) RT-PCR. (A-D) Expression of SLC24A5. (A) Expression initiates in the neural crest (arrow) and RPE at stage 25. (B) Expression continues in the melanophores as they mature from the neural crest as indicated by arrows. (C) Expression continues in the melanophores in the lateral pigmentation (LP) and increases in the RPE. (D) By stage 38 strong expression can be seen in melanophores of the lateral pigmentation, tail pigmentation (TP) and RPE. (E-H) Expression of the melanophore marker DCT in <i>X</i>.<i>laevis</i>. (E) Expression in the arising neural crest is indicated by arrow, (F) DCT is expressed in the melanophores as they mature from the neural crest as indicated by arrows. (G) and (H) Expression is maintained as melanophores migrate into lateral and tail pigmentation, expression continues in the RPE of the eye as indicated. (I-K) Histological analysis of SLC24A5 expression. (I) Section through the head reveals restricted expression to the RPE and dorsal head. (J) Section through the trunk shows expression in the flanks of the tadpole trunk and head. (K) whole embryo before sectioning, red line indicate approximate area where above sections were taken. (L) RT PCR analysis of SLC24A5 from various stages of <i>X</i>.<i>laevis</i> development. DCT was used a melanophores marker comparison, H4, loading control. Cells used were <i>X</i>.<i>laevis</i> melanophores (kind gift from Vladimir Gelfand).</p