Sterile Alpha Motif Containing 7 (Samd7) Is a Novel Crx-Regulated Transcriptional Repressor in the Retina

<div><p>Inherited retinal diseases are mainly caused by mutations in genes that are highly expressed in photoreceptors of the retina. The majority of these genes is under the control of the transcription factor Cone rod homeobox (Crx), that acts as a master transcription factor in photoreceptors. Using a genome-wide chromatin immunoprecipitation dataset that highlights all potential <i>in vivo</i> targets of Crx, we have identified a novel sterile alpha motif (SAM) domain containing protein, Samd7. mRNA Expression of Samd7 was confined to the late postnatal and adult mouse retina as well as the pineal gland. Using immunohistochemistry and Western blot, we could detect Samd7 protein in the outer nuclear layer of adult mouse retina. Ectopic over-expression in HEK293 cells demonstrated that Samd7 resides in the cytoplasm as well as the nucleus. <i>In vitro</i> electroporation of fluorescent reporters into living mouse retinal cultures revealed that transcription of the Samd7 gene depends on evolutionary conserved Crx motifs located in the first intron enhancer. Moreover, Crx knock-down with shRNA strongly reduced Samd7 reporter activity and endogenous Samd7 protein, indicating that Crx is required for retinal expression of Samd7. Finally, using co-transfections in luciferase reporter assays we found that Samd7 interferes with Crx-dependent transcription. Samd7 suppressed luciferase activity from a reporter plasmid with five Crx consensus repeats in a dose dependent manner and reduced Crx-mediated transactivation of regulatory sequences in the retinoschisin gene and the Samd7 gene itself. Taken together, we have identified a novel retinal SAM domain protein, Samd7, which could act as a transcriptional repressor involved in fine-tuning of Crx-regulated gene expression.</p> </div

Samd7 is expressed in the outer nuclear layer of the mouse retina and localizes to the nucleus of transfected cells.

<p>A: Immunhohistochemical analysis shows that Samd7 localizes to the outer nuclear layer in the adult mouse retina. Left panel: DAPI staining, right panel: anti-Samd7 antibody staining, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Scale bar, 50 µm. B: Western blot performed with retinal lysates detecting Samd7 at a molecular weight of 49 kDa and beta-actin as loading control. C: Western blot performed with protein lysates from naive HEK293 cells (Co) or HEK293 cells transfected with mock plasmid or Flag-Samd7 expression plasmid. Anti-Samd7 antibody, anti-flag antibody, and anti-beta-actin antibody were used. The Flag-Samd7 band had a molecular weight of approximately 51 kDa. D: Western blot of cytosolic and nuclear fractions of HEK293 cells transfected with mock plasmid or Flag-Samd7 expression vector. Samd7 was was detected in both the cytosolic and nuclear fractions at a molecular weight of 51 kDa. (E–P): Subcellular localization of Samd7 in HEK293 cells transfected with Flag-Samd7 expression vector shown in fluorescent Z-stacked optical images. Mock transfected cells did not show a specific red signal with either the anti-Flag (F) or the anti-Samd7 (L) antibody. The anti-Flag antibody (I, J) as well as the anti-Samd7 antibody (O, P) showed a specific nuclear staining in Flag-Samd7 transfected cells when couter-stained with DAPI.</p

Samd7 is a novel phylogenetically conserved SAM-domain protein.

<p>A: The full-length Samd7 protein comprises 445 amino acids including a 67 aa SAM domain, which is indicated by a box. B: Amino acid alignment of selected SAM domain sequences using Clustal W Blosum 62. The level of similarity is indicated by shading ranging from 100% (black) to less than 60% (light gray). C: Phylogenetic conservation of SAM-domain containing proteins. The branch length represents the number of substitutions that have ocurred in that branch and the distance scale represents the number of differences between sequences, with 0.1 meaning 10% difference between two sequences. D: Amino acid sequence similarities of mouse, rat, human, chicken, and zebrafish Samd7 proteins. The percentage of similarity is shown for the full-length protein as well as for individual regions of the protein, respectively.</p

Samd7 is expressed in the mouse retina and pineal gland.

<p>A: Relative mRNA expression of all SAM-domain containing proteins with isolated SAM domains in postnatal day 7 mouse retinas. Mean signal intensities of three independent Affymetrix mouse expression 430A arrays (GEO accession number GSE5581) show that Samd7 is the second most abundant transcript in the retina. B: RT-PCR analysis of total RNAs extracted from mouse stomach, lung, liver, testis, kidney, spleen, brain, retina, heart, and muscle reveals retina-specific mRNA expression of Samd7. C: RT-PCR analysis of total RNA extracted from mouse pineal gland. Primer pairs specific for Samd7, Samd11 and beta-actin were used for PCR. D: Real-time qRT-PCR analysis of early postnatal and adult retina demonstrates that Samd7 expression peaks at postnatal day 5 and then stays at intermediate levels.</p

Samd7 transcription is regulated by Crx.

<p>A: Identification of two Crx-bound regions (CBR1 and CBR2) at the mouse Samd7 locus. Enriched Crx ChIP-seq regions are shown in the proximal promoter and first intron of Samd7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060633#pone.0060633-Corbo1" target="_blank">[12]</a>. RNA polymerase II ChIP-Chip peaks at P2 and P25 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060633#pone.0060633-Tummala1" target="_blank">[32]</a> overlap with the Crx ChIP-Seq regions. The degree of mammalian conservation is indicated at the bottom. The nucleotide sequences of canonical Crx binding sequences (CBS) within CBR1 and CBR2 are depicted. B: The phylogenetic conservation of CBS1 and CBS2 within CBR2 is shown for several species. C-E: Activity of wild-type and mutant Samd7 CBRs in explanted mouse retinas. Co-electroporations were performed with pCAG-eGFP as control and the indicated Samd7 regulatory elements fused to dsRed. All constructs were electroporated at postnatal day 0 and the cultured explants were harvested at postnatal day 8. C: CBR1 is not active when fused to a promoterless dsRed reporter cassette. In contrast, CBR2 drives strong dsRed expression when fused to the minimal Rhodopsin promoter, which is <i>per se</i> not active. The cross-sections show that dsRed signals driven by Samd7 CBR2 were localized in the ONL, whereas the GFP signals by the ubiquitous control promoter were localized in the ONL, INL and GCL. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. D: Quantitative analysis of mutant constructs demonstrates that CBS1 and CBS3 are mandatory for high reporter expression of the intronic CBR2. E: Enhancer activity of CBR2 upstream of CBR1 also requires intact nucleotide sequences at CBS1 and CBS3.</p

Samd7-dependent suppression of Crx-activated promoters.

<p>A: Schematic drawing of retinoschisin, Samd7, and 5xCrx tk Luc reporter constructs used for luciferase assays in HEK293 cells. B: The 5xCrx-tk-Luc construct was co-transfected with Crx and various concentrations of Samd7 expression plasmids. C: Samd7 expression plasmid was co-transfected in the absence or presence of Crx vector and the retinoschisin promoter activity was determined. D: Samd7 expression plasmid was co-transfected in the absence or presence of Crx vector and the Samd7 promoter activity was determined. pSV β-galactosidase vector was co-transfected in each reaction to control for transfection efficiency. Error bars represent standard deviation of the mean (n = 6). **<i>P</i><0.01 and ***<i>P</i><0.001 One-way Analysis of Variance and Tukey's Post Hoc Test.</p