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

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.

Antagonistic regulation of Dlx2 expression by PITX2 and Msx2: implications for tooth development.

The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5'-TAATCC-3') with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.

The Pitx2 protein in mouse development.

The Rieger syndrome, an autosomal dominant disorder involving ocular, dental, and umbilical defects is caused by mutations in PITX2, a Bicoid-type homeobox protein. Mouse Pitx2 mRNA is expressed in eye, tooth and umbilicus consistent with the human Riegers phenotype. Moreover, Pitx2 is involved in the Nodal/Sonic hedgehog pathway that determines left/right polarity. In this report we demonstrate a 32-kDa polypeptide on Western blots of nuclear extracts from a rat pituitary cell line, using a Pitx2 specific antibody (designated P2R10). We describe also for the first time expression of the Pitx2 protein in mouse. Pitx2 protein immunostaining was detectable during the development of the eye, tooth, umbilicus, and also in the pituitary, heart, gut, and limb. We demonstrate for the first time directly that Pitx2 is asymmetrically expressed in early heart, gut, and lung development.

The human BARX2 gene: genomic structure, chromosomal localization, and single nucleotide polymorphisms.

The BARX genes 1 and 2 are Bar class homeobox genes expressed in craniofacial structures during development. In this report, we present the genomic structure, chromosomal localization, and polymorphic markers in BARX2. The gene has four exons, ranging in size from 85 to 1099 bp. BARX2 is localized on human chromosome 11q25, as determined by radiation hybrid mapping. In the mouse, Barx2 is coexpressed with Pitx2 in several tissues. Based on the coexpression, BARX2 was assumed to be a candidate gene for those cases of Rieger syndrome that cannot be associated with mutations of PITX2. Mutations in PITX2 cause some cases of Rieger syndrome, an autosomal dominant disorder affecting eyes, teeth, and umbilicus. DNA from Rieger patients was subjected to single-strand conformation polymorphism screening of the BARX2 coding region. Three single nucleotide polymorphisms were found in a normal population, although no etiologic mutations were detectable in over 100 cases of Rieger syndrome or in individuals with related ocular disorders.

A two unit antisense RNA cassette test system for silencing of target genes.

This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.

Bulged-out nucleotides protect an antisense RNA from RNase III cleavage.

Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem-loop of the antisense RNA, CopA, carry bulged-out nucleotides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced. The set of mutant and wild-type CopA variants was characterized structurally in vitro. The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. In vitro cleavage rates were drastically increased when either the lower or both bulges were absent. This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo. The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed. In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo.

Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo.

Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.