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Profiling of oxygen-modulated gene expression in early human placenta by systematic sequencing of suppressive subtractive hybridization products

¹ Génétique et Epigénétique des Pathologies Placentaires, GEPP, U709 Institut National de la Santé et de la Recherche Médicale-Université René Descartes-Institut Alfred Jost, Pavillon Baudelocque, Hôpital Cochin, Paris
² Centre National de la Recherche Scientifique UPR 9078-Université Paris V René Descartes, Hôpital Necker-Enfants Malades, Paris
³ Département de Physiologie Animale
⁴ Département de Génétique Animale, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy-en-Josas, France

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the "hypoxic" condition (2% O₂), whereas apoptotic genes are overrepresented in the "normoxic" condition (20% O₂). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O₂ tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.

hypoxia; genomic clustering; suppressive subtractive hybridization

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