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PITX2 regulates procollagen lysyl hydroxylase (PLOD) gene expression: implications for the pathology of Rieger syndrome.

Hjalt TA, Amendt BA, Murray JC - J. Cell Biol. (2001)

Bottom Line: Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements.The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments.Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242, USA.

ABSTRACT
The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.

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Clone B18 is immunoenriched by a Pitx2-specific antibody and hybridizes to single bands in a genomic Southern analysis. (A) The insert of clone B18 from the chromatin precipitation library was labeled and used to probe a filter with 30,000 plaques of nonamplified immunoenriched library. At least five strong signals are apparent and indicate an enrichment factor of 165. (B) Southern blot of total mouse DNA digested with PstI (lane 1) and EcoRI (lane 2).
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Figure 1: Clone B18 is immunoenriched by a Pitx2-specific antibody and hybridizes to single bands in a genomic Southern analysis. (A) The insert of clone B18 from the chromatin precipitation library was labeled and used to probe a filter with 30,000 plaques of nonamplified immunoenriched library. At least five strong signals are apparent and indicate an enrichment factor of 165. (B) Southern blot of total mouse DNA digested with PstI (lane 1) and EcoRI (lane 2).

Mentions: The nucleoprotein complexes of E14 mouse heads were immunoenriched by using a Pitx2-specific antibody (Hjalt and Murray 2000). The DNA was cloned into a phage library by plaque PCR using T7 and T3 flanking vector primers from 80 isolates; 90% contained inserts ranging in size from 100 to 8,000 bp, with an average size of 3,000 bp (not shown). 20 clones were excised at random one by one and inserts were labeled and used as probes for genomic Southern analyses and a plaque enrichment assay. One of the enriched clones, B18, that also represented a unique gene by Southern analysis (Fig. 1), displayed DNA sequence homology to mouse procollagen Plod-2. Normally, a 3,000-bp fragment of the mouse genome should appear 0.03 times on a 30,000 plaque filter (3E4 × 3E3/3E9). Five hits on the filter represent 165-fold enrichment. Control hybridization to a normal mouse genomic library filter of equal density was blank (not shown). The 2,701-bp B18 clone insert matched Plod-2 and PLOD-2 cDNA sequences in BLAST searches. Later we could confirm that B18 matched exon 7, intron 7, and exon 8 of PLOD-2. Since enrichment of the Plod-2 gene occurred before digestion with EcoRI and library cloning, we knew it would be possible to clone parts of the gene other than the promoter. We also confirmed that prolactin, a known Pitx2 binder (Amendt et al. 1998), was immunoenriched by probing a set of filters with a probe for mouse prolactin, exon 2 (not shown). Control hybridization to a normal mouse genomic library filter of equal density was blank (not shown). We also performed real-time PCR on normal total mouse DNA and immunoenriched DNA of the same concentrations. We used a primer set for 18S ribosomal DNA as a control and compared the results to those for the Plod-2 promoter, or exon 2 for prolactin. The normalized ΔCt values are presented in Table . The Plod-2 promoter was enriched 32-fold and the prolactin exon 2 was enriched 12-fold in this assay.


PITX2 regulates procollagen lysyl hydroxylase (PLOD) gene expression: implications for the pathology of Rieger syndrome.

Hjalt TA, Amendt BA, Murray JC - J. Cell Biol. (2001)

Clone B18 is immunoenriched by a Pitx2-specific antibody and hybridizes to single bands in a genomic Southern analysis. (A) The insert of clone B18 from the chromatin precipitation library was labeled and used to probe a filter with 30,000 plaques of nonamplified immunoenriched library. At least five strong signals are apparent and indicate an enrichment factor of 165. (B) Southern blot of total mouse DNA digested with PstI (lane 1) and EcoRI (lane 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196000&req=5

Figure 1: Clone B18 is immunoenriched by a Pitx2-specific antibody and hybridizes to single bands in a genomic Southern analysis. (A) The insert of clone B18 from the chromatin precipitation library was labeled and used to probe a filter with 30,000 plaques of nonamplified immunoenriched library. At least five strong signals are apparent and indicate an enrichment factor of 165. (B) Southern blot of total mouse DNA digested with PstI (lane 1) and EcoRI (lane 2).
Mentions: The nucleoprotein complexes of E14 mouse heads were immunoenriched by using a Pitx2-specific antibody (Hjalt and Murray 2000). The DNA was cloned into a phage library by plaque PCR using T7 and T3 flanking vector primers from 80 isolates; 90% contained inserts ranging in size from 100 to 8,000 bp, with an average size of 3,000 bp (not shown). 20 clones were excised at random one by one and inserts were labeled and used as probes for genomic Southern analyses and a plaque enrichment assay. One of the enriched clones, B18, that also represented a unique gene by Southern analysis (Fig. 1), displayed DNA sequence homology to mouse procollagen Plod-2. Normally, a 3,000-bp fragment of the mouse genome should appear 0.03 times on a 30,000 plaque filter (3E4 × 3E3/3E9). Five hits on the filter represent 165-fold enrichment. Control hybridization to a normal mouse genomic library filter of equal density was blank (not shown). The 2,701-bp B18 clone insert matched Plod-2 and PLOD-2 cDNA sequences in BLAST searches. Later we could confirm that B18 matched exon 7, intron 7, and exon 8 of PLOD-2. Since enrichment of the Plod-2 gene occurred before digestion with EcoRI and library cloning, we knew it would be possible to clone parts of the gene other than the promoter. We also confirmed that prolactin, a known Pitx2 binder (Amendt et al. 1998), was immunoenriched by probing a set of filters with a probe for mouse prolactin, exon 2 (not shown). Control hybridization to a normal mouse genomic library filter of equal density was blank (not shown). We also performed real-time PCR on normal total mouse DNA and immunoenriched DNA of the same concentrations. We used a primer set for 18S ribosomal DNA as a control and compared the results to those for the Plod-2 promoter, or exon 2 for prolactin. The normalized ΔCt values are presented in Table . The Plod-2 promoter was enriched 32-fold and the prolactin exon 2 was enriched 12-fold in this assay.

Bottom Line: Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements.The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments.Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242, USA.

ABSTRACT
The Rieger syndrome is an autosomal dominant disease characterized by ocular, craniofacial, and umbilical defects. Patients have mutations in PITX2, a paired-bicoid homeobox gene, also involved in left/right polarity determination. In this study we have identified a family of genes for enzymes responsible for hydroxylizing lysines in collagens as one group of likely cognate targets of PITX2 transcriptional regulation. The mouse procollagen lysyl hydroxylase (Plod)-2 gene was enriched for by chromatin precipitation using a PITX2/Pitx2-specific antibody. Plod-2, as well as the human PLOD-1 promoters, contains multiple bicoid (PITX2) binding elements. We show these elements to bind PITX2 specifically in vitro. The PLOD-1 promoter induces the expression of a luciferase reporter gene in the presence of PITX2 in cotransfection experiments. The Rieger syndrome causing PITX2 mutant T68P fails to induce PLOD-1-luciferase. Mutations and rearrangements in PLOD-1 are known to be prevalent in patients with Ehlers-Danlos syndrome, kyphoscoliosis type (type VI [EDVI]). Several of the same organ systems are involved in Rieger syndrome and EDVI.

Show MeSH
Related in: MedlinePlus