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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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The proximal promoter sequences of PLOD-1 and Plod-2. The proximal promoter DNA sequences of the human PLOD-1 (A) and mouse Plod-2 (B) are shown. PITX2 binding elements are in bold. The corresponding binding site names (A–J and A–H) are listed on the right side. The transcriptional start site of Plod-2 is not mapped, for reference purposes the 5′-most nucleotide of the cDNA (available from GenBank/EMBL/DDBJ under accession no. NM_011961) is used as +1. Bicoid-like and bicoid elements are in bold and boxed. In A, ApaI restriction sites are underlined. CAAT and TAATAA sequences are underlined with thick lines. These sequence data are available from GenBank/EMBL/DDBJ under accession nos. AF081786 (PLOD-1) and AF283255 (Plod-2).
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Figure 2: The proximal promoter sequences of PLOD-1 and Plod-2. The proximal promoter DNA sequences of the human PLOD-1 (A) and mouse Plod-2 (B) are shown. PITX2 binding elements are in bold. The corresponding binding site names (A–J and A–H) are listed on the right side. The transcriptional start site of Plod-2 is not mapped, for reference purposes the 5′-most nucleotide of the cDNA (available from GenBank/EMBL/DDBJ under accession no. NM_011961) is used as +1. Bicoid-like and bicoid elements are in bold and boxed. In A, ApaI restriction sites are underlined. CAAT and TAATAA sequences are underlined with thick lines. These sequence data are available from GenBank/EMBL/DDBJ under accession nos. AF081786 (PLOD-1) and AF283255 (Plod-2).

Mentions: Oligodeoxyribonucleotides (see Table and Fig. 2) were synthesized (Integrated DNA Technologies) in complementary pairs, annealed to form double stranded DNA, and labeled by kinasing with [γ-32P]ATP. The probes were purified on Sephacryl S-200 Microspin columns (Amersham Pharmacia Biotech) and the activity was measured by scintillation counting. Bacterially overexpressed PITX2 was prepared as described previously (Hjalt and Murray 2000). Electrophoretic mobility shift assays (EMSAs) were performed essentially as described in Amendt et al. 1998. The Plod-2 G probe was modified from the sequence in the promoter. The wild-type Plod-2 G probe would form concatemers during annealing (not shown). The GC-rich 3′ sequence (GCGCCGCGGG) was substituted for an arbitrary AT-rich sequence (AAAATCATGA).


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)

The proximal promoter sequences of PLOD-1 and Plod-2. The proximal promoter DNA sequences of the human PLOD-1 (A) and mouse Plod-2 (B) are shown. PITX2 binding elements are in bold. The corresponding binding site names (A–J and A–H) are listed on the right side. The transcriptional start site of Plod-2 is not mapped, for reference purposes the 5′-most nucleotide of the cDNA (available from GenBank/EMBL/DDBJ under accession no. NM_011961) is used as +1. Bicoid-like and bicoid elements are in bold and boxed. In A, ApaI restriction sites are underlined. CAAT and TAATAA sequences are underlined with thick lines. These sequence data are available from GenBank/EMBL/DDBJ under accession nos. AF081786 (PLOD-1) and AF283255 (Plod-2).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The proximal promoter sequences of PLOD-1 and Plod-2. The proximal promoter DNA sequences of the human PLOD-1 (A) and mouse Plod-2 (B) are shown. PITX2 binding elements are in bold. The corresponding binding site names (A–J and A–H) are listed on the right side. The transcriptional start site of Plod-2 is not mapped, for reference purposes the 5′-most nucleotide of the cDNA (available from GenBank/EMBL/DDBJ under accession no. NM_011961) is used as +1. Bicoid-like and bicoid elements are in bold and boxed. In A, ApaI restriction sites are underlined. CAAT and TAATAA sequences are underlined with thick lines. These sequence data are available from GenBank/EMBL/DDBJ under accession nos. AF081786 (PLOD-1) and AF283255 (Plod-2).
Mentions: Oligodeoxyribonucleotides (see Table and Fig. 2) were synthesized (Integrated DNA Technologies) in complementary pairs, annealed to form double stranded DNA, and labeled by kinasing with [γ-32P]ATP. The probes were purified on Sephacryl S-200 Microspin columns (Amersham Pharmacia Biotech) and the activity was measured by scintillation counting. Bacterially overexpressed PITX2 was prepared as described previously (Hjalt and Murray 2000). Electrophoretic mobility shift assays (EMSAs) were performed essentially as described in Amendt et al. 1998. The Plod-2 G probe was modified from the sequence in the promoter. The wild-type Plod-2 G probe would form concatemers during annealing (not shown). The GC-rich 3′ sequence (GCGCCGCGGG) was substituted for an arbitrary AT-rich sequence (AAAATCATGA).

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