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Kruppel-like factor 15, a zinc-finger transcriptional regulator, represses the rhodopsin and interphotoreceptor retinoid-binding protein promoters.

ABSTRACT: To identify novel transcriptional regulators of rhodopsin expression as a model for understanding photoreceptor-specific gene regulation.A bovine retinal cDNA library was screened in a yeast one-hybrid assay, with a 29-bp bovine rhodopsin promoter fragment as bait. Expression studies used RT-PCR and beta-galactosidase (LacZ) histochemistry of retinas from transgenic mice heterozygous for a targeted LacZ replacement of KLF15. Promoter transactivation assays measured luciferase expression in HEK293 cells transiently transfected with bovine rhodopsin or IRBP promoter-reporter constructs and expression constructs containing cDNAs for full or truncated KLF15, Crx (cone rod homeobox), and/or Nrl (neural retina leucine zipper). Data were analyzed with general linear models.The zinc-finger transcription factor KLF15 was identified as a rhodopsin-promoter-binding protein in a yeast one-hybrid screen. Expression was detected by RT-PCR in multiple tissues, including the retina, where KLF15-LacZ was observed in the inner nuclear layer, ganglion cell layer, and pigmented epithelial cells, but not in photoreceptors. KLF15 repressed transactivation of rhodopsin and IRBP promoters alone and in combination with the transcriptional activators Crx and/or Nrl. Repressor activity required both a 198-amino-acid element in the N-terminal domain and the C-terminal zinc finger DNA-binding domains.The zinc finger containing transcription factor KLF15 is a transcriptional repressor of the rhodopsin and IRBP promoters in vitro and, in the retina, is a possible participant in repression of photoreceptor-specific gene expression in nonphotoreceptor cells.

SUBMITTER: Otteson DC

PROVIDER: S-EPMC2660604 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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Kruppel-like factor 15, a zinc-finger transcriptional regulator, represses the rhodopsin and interphotoreceptor retinoid-binding protein promoters.

Otteson Deborah C DC Liu Yuhui Y Lai Hong H Wang ChenWei C Gray Susan S Jain Mukesh K MK Zack Donald J DJ

Investigative ophthalmology & visual science 20040801 8

<h4>Purpose</h4>To identify novel transcriptional regulators of rhodopsin expression as a model for understanding photoreceptor-specific gene regulation.<h4>Methods</h4>A bovine retinal cDNA library was screened in a yeast one-hybrid assay, with a 29-bp bovine rhodopsin promoter fragment as bait. Expression studies used RT-PCR and beta-galactosidase (LacZ) histochemistry of retinas from transgenic mice heterozygous for a targeted LacZ replacement of KLF15. Promoter transactivation assays measure ...[more]

PMID: 15277472

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Project description:Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

Project description:The interphotoreceptor retinoid-binding protein (IRBP) gene possesses an unusual structure, encoding multiple Repeats, each consisting of about 300 amino acids. Our goals were to gain insight into the function of IRBP, and to test the current model for the evolution of IRBP, in which Repeats were replicated from a simpler ancestral gene.We employed a bioinformatics approach to analyze IRBP loci in recently completed or near-complete genome sequences of several vertebrates and nonvertebrate chordates. IRBP gene expression in zebrafish was evaluated by reverse transcriptase PCR (RT-PCR) and in situ mRNA hybridizations with gene-specific probes.Patterns of exons and introns in the IRBP genes of tetrapods were highly similar, as were predicted amino acid sequences and Repeat structures. IRBP gene structure in teleost fish was more variable, and we report a new gene structure for two species, the Japanese puffer fish (Takifugu rubripes) and the zebrafish (Danio rerio). These teleost genomes contain a two-gene IRBP locus arranged head-to-tail in which the first gene, Gene 1, is intronless and contains a single large exon encoding three complete Repeats. It is followed by a second gene, Gene 2, which corresponds to the previously reported gene consisting of two Repeats spread across four exons and three introns. Each of the two zebrafish genes is transcribed. Gene 2 is expressed in the photoreceptors and RPE, and Gene 1 is expressed in the inner nuclear layer and weakly in the ganglion cell layer.The tetrapod IRBP gene structure is highly conserved while the teleost fish gene structure was a surprise: It appears to be a two-gene locus with distinct Repeat organization in each open reading frame. This gene structure and gene expression data are consistent with possible neofunctionalization or sub-function partitioning of Gene 1 and Gene 2 in the zebrafish. We suggest that the two-gene locus in teleost fish arose as a consequence of either the known whole genome duplication or single gene tandem duplication.

Project description:BackgroundIn the retina, many of the genes that encode components of the visual transduction cascade and retinoid recycling are exclusively expressed in photoreceptor cells and show highly stereotyped temporal and spatial expression patterns. Multiple transcriptional activators of photoreceptor-specific genes have been identified, but little is known about negative regulation of gene expression in the retina. We recently identified KLF15, a member of the Sp/Krüppel-like Factor family of zinc-finger containing transcription factors, as an in vitro repressor of the promoters of the photoreceptor-specific genes rhodopsin and IRBP/Rbp3. To gain further insight into the mechanism of KLF15-mediated regulation of gene expression, we have characterized the binding characteristics and specificity of KLF15's DNA binding domains and defined the KLF15 binding sites in the rhodopsin and IRBP promoters.ResultsIn EMSA and DNAseI footprinting assays, a KLF15-GST fusion protein containing the C-terminal zinc-finger domains (123 amino acids) showed zinc-dependent and sequence-specific binding to a 9 bp consensus sequence containing a core CG/TCCCC. Both the bovine rhodopsin and IRBP promoters contained multiple KLF15 binding sites that included the previously identified CRS-1 and G-rich repressor elements. KLF15 binding sites were highly conserved between the bovine, human, chimp and dog rhodopsin promoters, but less conserved in rodents. KLF15 reduced luciferase expression by bRho130-luc (containing 4 KLF15 sites) and repressed promoter activation by CRX (cone rod homeobox) and/or NRL (neural retina leucine zipper), although the magnitude of the reduction was smaller than previously reported for a longer bRho225-luc (containing 6 KFL15 sites).ConclusionKLF15 binds to multiple 9 bp consensus sites in the Rhodospin and IRBP promoters including the CRS-1 and G-rich repressor elements. Based on the known expression pattern of KLF15 in non-photoreceptor cells, we hypothesize an in vivo role for KLF15 in repressing photoreceptor-specific gene expression in the inner retina.

Dataset Information

Kruppel-like factor 15, a zinc-finger transcriptional regulator, represses the rhodopsin and interphotoreceptor retinoid-binding protein promoters.

Publications

Kruppel-like factor 15, a zinc-finger transcriptional regulator, represses the rhodopsin and interphotoreceptor retinoid-binding protein promoters.

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