Institute of Human Genetics, University of Regensburg, Regensburg, Germany.
PLoS One. 2013;8(4):e60633. doi: 10.1371/journal.pone.0060633. Epub 2013 Apr 2.
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 in vivo 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. In vitro 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.
遗传性视网膜疾病主要是由高度表达在视网膜感光细胞中的基因突变引起的。这些基因的大多数都受转录因子 Cone rod homeobox (Crx)的控制，它是感光细胞中的主要转录因子。我们利用全基因组染色质免疫沉淀数据集，该数据集突出了 Crx 的所有潜在体内靶标，鉴定了一种新的含有 sterile alpha motif (SAM)结构域的蛋白 Samd7。Samd7 的 mRNA 表达局限于新生后和成年期的小鼠视网膜以及松果体。通过免疫组织化学和 Western blot，我们可以在成年期小鼠视网膜的外核层检测到 Samd7 蛋白。HEK293 细胞中的异位过表达表明 Samd7 存在于细胞质和细胞核中。在活体小鼠视网膜培养物中用电穿孔将荧光报告基因转染后，发现 Samd7 基因的转录依赖于位于第一内含子增强子中的进化保守的 Crx 基序。此外，用 shRNA 敲低 Crx 强烈降低了 Samd7 报告基因的活性和内源性 Samd7 蛋白，表明 Crx 是 Samd7 在视网膜表达所必需的。最后，我们在荧光素酶报告基因检测中发现，Samd7 干扰 Crx 依赖性转录。Samd7 以剂量依赖的方式抑制来自具有五个 Crx 共有重复的报告质粒的荧光素酶活性，并降低了视蛋白基因和 Samd7 基因自身中 Crx 介导的调节序列的转激活作用。总之，我们鉴定了一种新的视网膜 SAM 结构域蛋白 Samd7，它可能作为一种转录抑制剂参与精细调节 Crx 调节的基因表达。
