Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-I and G-rich repressor elements

Deborah C. Otteson, Hong Chen Lai, Yuhui Liu, Donald J Zack

Research output: Contribution to journalArticle

Abstract

Background: In 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. Results: In 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). Conclusion: KLF15 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.

Original languageEnglish (US)
Article number15
JournalBMC Molecular Biology
Volume6
DOIs
StatePublished - Jun 17 2005

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Rhodopsin
Zinc Fingers
Retina
Zinc
Gene expression
Genes
Binding Sites
Gene Expression Regulation
Gene Components
Leucine Zippers
Photoreceptor Cells
Vertebrate Photoreceptor Cells
Homeobox Genes
Retinoids
Consensus Sequence
Recycling
Protein C
Luciferases
Cones
Rodentia

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Molecular Biology

Cite this

@article{bee5073c669d42b5bc545ff392578777,
title = "Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-I and G-rich repressor elements",
abstract = "Background: In 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/Kr{\"u}ppel-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. Results: In 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). Conclusion: KLF15 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.",
author = "Otteson, {Deborah C.} and Lai, {Hong Chen} and Yuhui Liu and Zack, {Donald J}",
year = "2005",
month = "6",
day = "17",
doi = "10.1186/1471-2199-6-15",
language = "English (US)",
volume = "6",
journal = "BMC Molecular Biology",
issn = "1471-2199",
publisher = "BioMed Central",

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TY - JOUR

T1 - Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-I and G-rich repressor elements

AU - Otteson, Deborah C.

AU - Lai, Hong Chen

AU - Liu, Yuhui

AU - Zack, Donald J

PY - 2005/6/17

Y1 - 2005/6/17

N2 - Background: In 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. Results: In 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). Conclusion: KLF15 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.

AB - Background: In 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. Results: In 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). Conclusion: KLF15 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.

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