Hydra is a diploblastic animal which presents exceptional faculties of regeneration. Whatever its age, the animal repairs the part of the body cut down, this process of regeneration corresponding to the reactivation of a program of development. Repairing events depend on the ability to eliminate the most damaged cells and to protect the others from apoptotic and autophagic events. Thanks to the RNA interference mechanism that efficiently silence gene expression when hydra are fed with double- stranded RNAs (dsRNAs), we could analyze the cellular alterations that occur when genes that are activated immediately or early after amputation are knocked-down. During this master stage, my contribution focused on three distinct aspects, firstly on the characterization of the CREB-binding protein (CBP) coding unit, secondly on the identification of the alterations observed in Kazal1(RNAi), RSK(RNAi), CREB(RNAi) and CBP(RNAi) cells from regenerating hydra, thirdly on the characterization of reliable tools to detect histone modifications in regenerating tips.
The reactivation of the developmental program depends on the ability of the adult tissues to reorganize. This reorganisation implies the activation of specific genes that will control different cellular processes like cell proliferation and apoptosis. Among these genes, we focused on those interacting with the cAMP Response Element Binding protein (CREB). CREB is an evolutionarily-conserved transcription factor that was previously shown to get phosphorylated during early head regeneration; .the kinase responsible for CREB phosphorylation being the Ribosomal S6 Kinase (RSK). In fact pharmacological inhibition of RSK activity prevented head regeneration suggesting that RSK-dependent phosphorylation is essential for head regeneration. As in bilaterians, we expected the phosphorylated CREB to interact with the co-activator protein CBP (CREB Binding Protein) to modulate gene transcription. CBP is a large (~300 kD), multifunctional protein, highly conserved during evolution, which controls gene expression by chromatin modifications due to its Histone Acetyling Transferase (HAT) activity. CBP acts as a transcriptional co-activator, interacting with a large variety of nuclear proteins including the phosphorylated form of the CREB transcription factor. It is present in many animal species and was also detected in plants where the CREB interacting domain called “KIX”, identified in the vertebrate CBPs, is totally missing, but the HAT domain is well conserved and active.
My project was to focus on CBP at the gene, protein and activity levels. Some results were already available: two partial cDNA fragments covering the C-moiety were characterized and some data about the mRNA and protein expression patterns were available. It was shown that CBP mRNA expression was enhanced in regenerating tips and that an antibody selected to target the KIX domain of the mouse CBP cross- reacted on Hydra cells. Moreover an anti-acetylated histone-H4 antibody had shown a strong fluorescent signal in regenerating tips. My job was to continue the project, at first I had to clone the KIX and HAT domains of the Hydra CBP gene as the KIX domain had not yet been proved to exist in cnidarians. That step was successful; a great proportion of the gene was isolated, sequenced. The deduced sequences of the hydra KIX and HAT domains showed a high level of conservation in phylogenetic analyses.
