Prx1 and Prx2 are upstream regulators of sonic hedgehog and control cell proliferation during mandibular arch morphogenesis.

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. We investigated development of the mandibular arch of Prx1(-/-)Prx2(-/-) mutants to obtain insight into the molecular basis of the lower jaw abnormalities. We observed in mutant embryos a local decrease in proliferation of mandibular arch mesenchyme in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression was strongly reduced, indicative of a function for Prx genes in indirect regulation of SHH: Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopied the lower jaw defects of Prx1(-/-)Prx2(-/-) mutants. In addition, this treatment led to loss of the mandibular incisors. We present a model that describes how loss of Shh expression in Prx1(-/-)Prx2(-/-) mutants leads to abnormal morphogenesis of the mandibular arch.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements.

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Polycomb and bmi-1 homologs are expressed in overlapping patterns in Xenopus embryos and are able to interact with each other.

The Polycomb group genes in Drosophila are involved in the stable and inheritable repression of gene expression. The Polycomb group proteins probably operate as multimeric complexes that bind to chromatin. To investigate molecular mechanisms of stable repression of gene activity in vertebrates we have begun to study Xenopus homologs of Polycomb group genes. We identified the Xenopus homologs of the Drosophila Polycomb gene and the bmi-1 gene. bmi-1 is a proto-oncogene which has sequence homology with the Polycomb group gene Posterior Sex Combs. We show that the XPolycomb and Xbmi-1 genes are expressed in overlapping patterns in the central nervous system of Xenopus embryos. However, XPolycomb is also expressed in the somites, whereas Xbmi-1 is not. We further demonstrate that the XPolycomb and Xbmi-1 proteins are able to interact with each other via conserved sequence motifs. These data suggest that also vertebrate Polycomb group proteins form multimeric complexes.

Haemophilia B Leyden: the effect of mutations at position +13 on the liver-specific transcription of the factor IX gene.

Haemophilia B Leyden is characterized by low plasma levels (< or = 1-13% of normal) of blood coagulation factor IX during childhood. After the onset of puberty, plasma factor IX levels gradually rise, probably under the influence of androgens. Single point mutations have been detected in the factor IX promoter at -21, -20, -6, -5, +6, +8 and +13. This paper examines how two of these mutations (a deletion of an A, and an A-->G substitution at +13) interfere with normal factor IX gene transcription. It is shown that both mutations do impair factor IX promoter activity in transiently transfected HepG2 cells. The mutations at +13 lie in a region (+1 to +18) that is considered to contain a binding site for the CCAAT/enhancer binding protein. Transactivation by the CCAAT/enhancer binding protein alpha of the wild-type and mutated factor IX promoter (-192 to +38) resulted in an approximately four-fold and approximately two-fold, respectively, increase of CAT activity. Gel mobility shift assays revealed that the binding of the CCAAT/enhancer binding protein alpha is disrupted by both the deletion of an A, and the A-->G substitution at +13. The role of two additional bZIP factors, D-site binding protein and liver-enriched transcriptional activator protein, in the binding and activation of the +13 factor IX promoter region was examined. The D-site binding protein binds to the factor IX promoter region (+1 to +18) in gel mobility shift assays. The deletion of an A at +13 does not interfere with the binding of the D-site binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemophilia B Leyden: substitution of thymine for guanine at position -21 results in a disruption of a hepatocyte nuclear factor 4 binding site in the factor IX promoter.

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by an altered developmental expression of blood coagulation factor IX. This form of hemophilia B has been found to be associated with a variety of single point mutations in the factor IX promoter region. We now describe a novel point mutation, T-->G at position -21, in two related patients with the hemophilia B Leyden phenotype. This mutation lies within the factor IX promoter region (-40 to -9) that contains overlapping binding sites for hepatocyte nuclear factor 4 (HNF-4) and androgen receptor. Transient transfection assays in HepG2 cells show that the -21 mutation causes a significant reduction in factor IX promoter activity. Gel mobility shift assays and transient cotransfection experiments revealed that the HNF-4-binding site but not the androgen-responsive element is disrupted by the -21 mutation. A comparison of the -21 mutation with the previously described -20 T-->A mutation (associated with the hemophilia B Leyden phenotype) and -26 G-->C mutation (associated with severe hemophilia B throughout life) was made. It shows that the -21 mutation reduced HNF-4 binding and transactivation to a similar level as the -20 mutation, whereas the -26 mutation completely abolished HNF-4 binding and transactivation. Mobility shift experiments indicate that there was no significant difference in binding affinity of recombinant androgen receptor protein for oligonucleotides containing wild-type and -21 or -20 mutated DNA. The binding affinity for the oligonucleotide containing the -26 mutation was twofold lower. The results indicate that the disruption of the HNF-4-binding site by the -21 T-->G mutation is the cause of the bleeding disorder in these two patients. This study adds further support for the notion that the recovery from hemophilia at puberty may not only be related to an intact androgen-responsive element but also to the degree of disruption of the HNF-4-binding site.

Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden.

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by very low plasma levels of blood coagulation factor IX (fIX) during childhood. After puberty, plasma fIX levels gradually rise to a maximum of 60% of normal, probably under the influence of testosterone. Single point mutations in the fIX promoter region of hemophilia B Leyden patients have been reported at -20, -6, -5, +8 and +13. In addition, one promoter mutation (G----C at -26) has been detected that abolishes fIX expression throughout life (M. Ludwig, personal communication). We examined how one of the hemophilia B Leyden mutations (T----A at -20) and the G----C mutation at -26 interfere with fIX gene transcription. We report that the wild-type promoter of the human fIX gene contains a binding site (at nucleotides -34 to -10) for hepatocyte nuclear factor 4 (HNF-4), a member of the steroid hormone receptor superfamily of transcription factors. The binding of HNF-4 is disrupted by both the T----A mutation at -20 and the G----C mutation at -26. Whereas HNF-4 transactivates the wild-type promoter sequence in liver (HepG2) and non-liver (HeLa) cell types quite well, it transactivates the -20 mutated promoter to only a limited extent and the -26 mutated promoter not at all. These data suggest that HNF-4 is a major factor controlling fIX expression in the normal individual and that its inability to bind efficiently to the -20 T----A and the -26 G----C mutated promoter sequence results in hemophilia. Further, the severity of the hemophilia phenotype appears to be directly related to the degree of disruption of HNF-4 binding and transactivation.

Localization of transcription initiation sites in the human coagulation factor IX gene.

The transcription start sites of the human gene coding for the coagulation factor IX have been identified. Three major transcription initiation sites within a small area of approximately 30 nucleotides were found by S1 nuclease analysis and primer extension studies.