Monique Frain

The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain

The nuclear protein LF-B1 (also referred to as HNF-1) is a transcription activator required for the expression of several liver-specific genes. LF-B1 has been purified to homogeneity from rat liver nuclear extracts. The sequence of the protein has been partially determined and, subsequently, overlapping cDNA clones containing the entire open reading frame of LF-B1 were isolated. The full-length cDNA encodes a 628 amino acid protein and directs the synthesis in vitro of a protein capable of binding DNA with the same specificity as LF-B1. The cDNA was recombined into a vaccinia virus vector and active LF-B1 was obtained from infected HeLa cells. Addition of the vaccinia recombinant protein to rat spleen extracts results in activation of transcription of an LF-B1-dependent promoter. The DNA binding domain of LF-B1 is located in the amino-terminal part of the protein and displays distant structural similarity to the homeobox domain. The distribution of LF-B1 mRNA is restricted to liver, which correlates with the tissue-specific expression of its target genes.

The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation

The zinc finger gene Krox20 and many Hox homeobox genes are expressed in segment-restricted domains in the hindbrain. The restricted expression patterns appear before morphological segmentation, suggesting that these transcription factors may play an early role in the establishment and identity of rhombomeric segments. In this paper, we show that the HoxB2 (Hox2.8) gene is normally upregulated in rhombomeres (r) 3, 4, and 5, and we identify an enhancer region upstream of the gene that imposes r3/r5 expression in transgenic mice. This enhancer contains three Krox20-binding sites required in vitro for complex formation with Krox20 protein and in vivo for rhombomere-restricted expression. In transgenic mice, Krox20 expressed in ectopic domains can transactivate a reporter construct containing the HoxB2 r3/r5 enhancer. These data demonstrate that Krox20 is a part of the upstream transcriptional cascade that directly regulates HoxB2 expression during hindbrain segmentation.

N-Cadherin Interacts with Axin and LRP5 To Negatively Regulate Wnt/β-Catenin Signaling, Osteoblast Function, and Bone Formation

ABSTRACT Wnt signaling plays an important role in the regulation of bone formation and bone mass. The mechanisms that regulate canonical Wnt signaling in osteoblasts are not fully understood. We show here a novel mechanism by which the adhesion molecule N-cadherin interacts with the Wnt coreceptor LRP5 and regulates canonical Wnt/β-catenin signaling in osteoblasts. We demonstrate that N-cadherin, besides associating with β-catenin at the membrane, forms a molecular complex with axin and LRP5 involving the LRP5 cytoplasmic tail domain. N-cadherin overexpression in osteoblasts increases N-cadherin-LRP5 interaction, causing increased β-catenin degradation and altered TCF/LEF transcription in response to Wnt3a. This mechanism results in decreased osteoblast gene expression and osteogenesis in basal conditions and in response to Wnt3a. Consistent with a functional mechanism, silencing N-cadherin expression in control cells increases TCF/LEF transcription and enhances the response to Wnt3a. Using N-cadherin transgenic mice, we show that increased N-cadherin-LRP5 interaction resulting from targeted overexpression of N-cadherin in osteoblasts causes increased β-catenin ubiquitination and results in cell-autonomous defective osteoblast function, reduced bone formation, and delayed bone mass acquisition. These data indicate that a previously unrecognized N-cadherin-axin-LRP5 interaction negatively regulates Wnt/β-catenin signaling and is critical in the regulation of osteoblast function, bone formation, and bone mass.

Differential expression of albumin and α-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.

Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control.

Neural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning.

Krox20 hindbrain cis-regulatory landscape: interplay between multiple long-range initiation and autoregulatory elements.

The vertebrate hindbrain is subject to a transient segmentation process leading to the formation of seven or eight metameric territories termed rhombomeres (r). This segmentation provides the basis for the subsequent establishment of hindbrain neuronal organization and participates in the patterning of the neural crest involved in craniofacial development. The zinc-finger gene Krox20 is expressed in r3 and r5, and encodes a transcription factor that plays a key role in hindbrain segmentation, coordinating segment formation, specification of odd- and even-numbered rhombomeres, and cell segregation between adjacent segments, through the regulation of numerous downstream genes. In order to further elucidate the genetic network underlying hindbrain segmentation, we have undertaken the analysis of the cis-regulatory sequences governing Krox20 expression. We have found that the control of Krox20 transcription relies on three very long-range (200 kb) enhancer elements (A, B and C) that are conserved between chick, mouse and human genomes. Elements B and C are activated at the earliest stage of Krox20 expression in r5 and r3-r5, respectively, and do not require the Krox20 protein. These elements are likely to function as initiators of Krox20 expression. Element B contains a binding site for the transcription factor vHNF1, the mutation of which abolishes its activity, suggesting that vHNF1 is a direct initiator of Krox20 expression in r5. Element A contains Krox20-binding sites, which are required, together with the Krox20 protein, for its activity. This element therefore allows the establishment of a direct positive autoregulatory loop, which takes the relay of the initiator elements and maintains Krox20 expression. Together, our studies provide a basis for a model of the molecular mechanisms controlling Krox20 expression in the developing hindbrain and neural crest.

Nucleotide sequence of a cDNA clone for human aldolase B

Two specific clones for human aldolase B were isolated from a human liver cDNA library using a rat aldolase B cDNA probe. The clones were identified by positive hybridization-selection and one of them was sequenced. The 127 C-terminal residues of the human protein were deduced from this nucleotide sequence analysis. They showed 92% homology with the corresponding previously published amino-acid sequence of rat liver aldolase B.

Analysis of fibrinogen genes in patients with congenital afibrinogenemia.

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.

Human Inter-alpha-Trypsin-Inhibitor: Characterization and partial nucleotide sequencing of a light chain-encoding cDNA

A synthetic Inter-alpha-Trypsin-Inhibitor (ITI) -specific oligonucleotide probe was used to isolate a clone from a human liver cDNA library. The amino-acid sequence deduced from partial nucleotide sequencing of the corresponding cDNA insert perfectly matched a known ITI sequence, apart from an as yet unreported C-terminal dipeptide. Hybridization on Northern blots evidenced that this cDNA insert originated from an ITI light chain-encoding mRNA whose size was estimated to be 1 300 bases.

Immunological screening of standard cDNA libraries in pBR322 vectors: Detection of human fibrinogen and prothrombin cDNA clones

The in situ immunological detection of antigens encoded by cDNA inserted into the PstI site of pBR322 plasmids was optimized. It was found that sensitivity of the detection was dramatically increased by in situ amplification of the recombinant plasmids on chloramphenicol-containing medium followed by a brief incubation without chloramphenicol during which protein synthesis resumes. In addition, several modifications of the previously described methods which permit total suppression of background and false positives are described. These techniques allowed easy detection of cDNA clones for human B beta- and gamma-fibrinogen and -prothrombin using a human liver double-stranded cDNA recombinant plasmid library in pBR322 vectors.