Denise Aragnol

Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control

Pontin (Pont) and Reptin (Rept) are paralogous ATPases that are evolutionarily conserved from yeast to human. They are recruited in multiprotein complexes that function in various aspects of DNA metabolism. They are essential for viability and have antagonistic roles in tissue growth, cell signalling and regulation of the tumour metastasis suppressor gene, KAI1, indicating that the balance of Pont and Rept regulates epigenetic programmes critical for development and cancer progression. Here, we describe Pont and Rept as antagonistic mediators of Drosophila Hox gene transcription, functioning with Polycomb group (PcG) and Trithorax group proteins to maintain correct patterns of expression. We show that Rept is a component of the PRC1 PcG complex, whereas Pont purifies with the Brahma complex. Furthermore, the enzymatic functions of Rept and Pont are indispensable for maintaining Hox gene expression states, highlighting the importance of these two antagonistic factors in transcriptional output.

dlarp, a new candidate Hox target in Drosophila whose orthologue in mouse is expressed at sites of epithelium/mesenchymal interactions.

Hox complex genes are key developmental regulators highly conserved throughout evolution. They encode transcription factors that initiate genetic programs of diversified morphogenesis along the anteroposterior embryonic axis. We report the characterization of the novel Drosophila Hox target gene dlarp, isolated from a further screen of a previously described library of genomic DNA fragments associated in vivo with Ultrabithorax proteins. The dlarp spatio‐temporal pattern of transcription in wild‐type and homeotic mutant embryos is consistent with a positive regulation by Sex combs reduced and Ultrabithorax in the parasegment 2 ectoderm and the abdominal mesoderm, respectively. The teashirt gene product, thought to act in concert with Hox proteins, is also required for the transcriptional control of this target. Search in databases revealed that dlarp has been highly conserved during evolution. The embryonic expression pattern of the mouse orthologue does not support a function downstream of Hox proteins. It is mainly transcribed in neural structures and in developing organs characterized by epithelial‐mesenchymal interactions. Dev Dyn 2000;218:401–413.

nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis

From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.

Effect of ligand binding and of pH change on the accessibility of thiol residues of fructose-1,6-bisphosphatase from spinach chloroplast

Sulfhydryl groups of reduced fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) from spinach chloroplast have been classified in several sets of different accessibility according to their rate of reaction with dithiobis(nitrobenzoic acid). The oxidation by this reagent results from the linkage of vicinal thiols into disulfide bridges. The thiol groups involved in the activation-deactivation process have been characterized by analyzing the effects of a stepwise oxidation on the catalytic activity of the reduced enzyme. The target of the redox activation process is constituted by two disulfide bridges as indicated by the fact that the four resulting thiols are essential residues for the activity. Mg2+ or substrate fixation modifies the enzyme conformation and the accessibility of the strategic sulfhydryls. O-Methylfructofuranoside 1,6-bisphosphate, a non-reactive substrate analog, induces the same conformational transition as the one induced by the substrate. The simultaneous fixation of Mg2+ and the substrate analog maintains the reduced enzyme in a specific conformation which is very sensitive to pH variations. At pH 7 the four strategic thiol groups are deeply embedded in the protein but are directly exposed to oxidation at pH 8.

Analysis of paralogous pontin and reptin gene expression during mouse development

Evolutionarily conserved from yeast to human, the paralogous DNA helicases Pontin (Pont) and Reptin (Rept) are simultaneously recruited in multi-protein chromatin complexes that function in different aspects of DNA metabolism (transcription, replication and repair). When assayed, the two proteins were found to be essential for viability and to play antagonistic roles, suggesting that the balance of Pont/Rept regulates epigenetic programmes critical for development. Consistent with this, the two helicases are provided in the same embryonic territories during Drosophila development. In Xenopus, while transcribed in the same regions early in embryogenesis, pont and rept adopt significantly different patterns afterwards. Here we report that the two genes follow highly resembling transcription patterns in mouse embryos, with prominent expression in limb buds and branchial arches, organs undergoing mesenchymal–epithelial interactions and in motoneurones from cranial and spinal regions. Thus, simultaneous expression during development appears to constitute another feature of the evolutionary conservation of pont and rept genes.

Effect of ligand binding on the pH-induced conformational change of fructose-1,6-bisphosphatase from spinach chloroplast

The conformational transitions of reduced fructose-1,6-bisphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from spinach chloroplast, which occur upon raising the pH from 7 to 8 or, inversely, decreasing it from 8 to 7, have been analyzed spectrometrically by monitoring the induced fluorescence changes either of an extrinsic fluorescent probe or of intrinsic tryptophan residues. A simple model is presented to relate the catalytic activity to the conformation state of the reduced enzyme. Upon pH increase and ionization of a strategic group the inactive enzyme undergoes a fast conformational change and is then slowly converted into an active state. These conformation changes are fully reversible. Two parameters of the slow relaxation, the observed rate constant and the amplitude, have been analyzed as a function of the proton concentration in the absence of ligand, in the presence of substrate, in the presence of Mg2+ and also in the presence of Mg2+ and of a substrate analog. When the reduced enzyme has bound Mg2+, the observed rate constant of the slow relaxation is identical to that of catalytic activity. The results obtained suggest that the pH-induced conformational change is the rate-limiting step in the overall activation of fructose-1,6-bisphosphatase.