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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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PITX2 upregulates PLOD-1–luciferase fusion constructs. The PLOD-1 promoter was fused to a luciferase reporter gene and assayed for activation by PITX2A. (A) CHO cells. (B) HeLa cells.
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Figure 4: PITX2 upregulates PLOD-1–luciferase fusion constructs. The PLOD-1 promoter was fused to a luciferase reporter gene and assayed for activation by PITX2A. (A) CHO cells. (B) HeLa cells.

Mentions: We proceeded to clone the PLOD-1 promoter in a luciferase reporter gene vector. We characterized the response to PITX2 by cotransfecting the PLOD-1–luciferase construct with a PITX2 expression vector. In CHO cells, the PLOD-1 261 construct containing 10 bicoid elements was activated 20-fold by PITX2A (Fig. 4 A). The minimal promoter constructs, PLOD-1 2561, were modestly activated (approximately fivefold) by PITX2. We attribute the modest activation to the fact that two bicoid-like sites remain, one TAACCC and one TAAGCC, in the minimal construct. Such elements are known to bind PITX2 in vitro (Amendt, B., unpublished data). In HeLa cells, PLOD-1 was activated 6.5-fold by PITX2A (Fig. 4 B). Furthermore, we found that a PITX2 gene carrying the Rieger syndrome causing mutation T68P failed to upregulate PLOD-1 in cell culture (Fig. 5). This protein can be expressed in cell culture (Amendt et al. 1998). The responses were similar to the negative controls both in CHO cells (Fig. 5 B) and in HeLa cells (Fig. 5 C).


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)

PITX2 upregulates PLOD-1–luciferase fusion constructs. The PLOD-1 promoter was fused to a luciferase reporter gene and assayed for activation by PITX2A. (A) CHO cells. (B) HeLa cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196000&req=5

Figure 4: PITX2 upregulates PLOD-1–luciferase fusion constructs. The PLOD-1 promoter was fused to a luciferase reporter gene and assayed for activation by PITX2A. (A) CHO cells. (B) HeLa cells.
Mentions: We proceeded to clone the PLOD-1 promoter in a luciferase reporter gene vector. We characterized the response to PITX2 by cotransfecting the PLOD-1–luciferase construct with a PITX2 expression vector. In CHO cells, the PLOD-1 261 construct containing 10 bicoid elements was activated 20-fold by PITX2A (Fig. 4 A). The minimal promoter constructs, PLOD-1 2561, were modestly activated (approximately fivefold) by PITX2. We attribute the modest activation to the fact that two bicoid-like sites remain, one TAACCC and one TAAGCC, in the minimal construct. Such elements are known to bind PITX2 in vitro (Amendt, B., unpublished data). In HeLa cells, PLOD-1 was activated 6.5-fold by PITX2A (Fig. 4 B). Furthermore, we found that a PITX2 gene carrying the Rieger syndrome causing mutation T68P failed to upregulate PLOD-1 in cell culture (Fig. 5). This protein can be expressed in cell culture (Amendt et al. 1998). The responses were similar to the negative controls both in CHO cells (Fig. 5 B) and in HeLa cells (Fig. 5 C).

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