Le rôle des ‘Nucleosome Assembly Proteins’ dans les cellules souches neuronales et les neurones – NEURONAP

The X-linked gene Nap1l2/NAP1L2 encodes a protein that shows similarities to the nucleosomal assembly (NAP) and SET proteins. The gene is specifically expressed in nestin positive neural stem cells and neurons, with targeted deletion of the murine gene leading to embryonic lethality associated with spina bifida and exencephaly. This phenotype is associated ex vivo and in vivo with a modification of stem cell renewal/differentiation pathway of neural stem cells. Whilst the precise mechanisms underlying these phenotypes remain unclear NAP1L2 has been shown to associate with chromatin and to interact with histones H3, H4. Loss of NAP1L2 results in decreased histone acetylation activity and is associated with important transcriptional changes in differentiating neurons, which include the marked downregulation of the Cyclin dependent kinase inhibitor 1c (Cdkn1c) gene. Cdkn1c expression normally increases during neuronal differentiation and this correlates with the specific recruitment of the NAP1L2 protein at the site of Cdkn1c transcription. These results have led us to suggest that the NAP1L2 protein plays an important role in regulating transcription in developing neurons via the control of histone modification. Our data support the idea that neuronal NAPs mediate cell-type specific mechanisms of establishment/modification of a chromatin permissive state that can affect neurogenesis and neuronal survival. This finding opens up interesting experimental possibilities for modulating the ex vivo production and expansion of stem cells of the neuronal lineage. The availability of such neuronal stem cells and their derivatives is likely to be of major therapeutic importance for many types of neurological and neuromuscular aetiologies. A better understanding of the mode of action of the mouse NAP1L2 at the molecular level and of its interactive partners could impact, through the exploitation of its human homologue, the manipulation of human neural stem cell proliferation and differentiation. In this context we propose to study the mechanisms of action of the Nucleosome Assembly Protein 1-like 2 (NAP1L2) gene and the role of its immediate partners in the control of murine neural stem cell proliferation and differentiation. Experiments will be undertaken to: 1) Complete the identification of NAP1L2 target genes .This objective will be attained by comparative transcriptional profiling of wild type nestin positive and neuronal cells with their counterparts mutated for Nap1l2. Conditional deletion will allow the hierarchy of candidate genes to be explored. 2) Define the proteins interacting with NAP1L2 to ensure its action. We will build on previous two-hybrid approaches to define fully the complexes involving NAP1L2 by a proteomics approach and their mode of action. Tissue specificity of these complexes in neural stem cells and mature neurones will be explored. 3) In so far as possible candidate sequences revealed by the two approaches will be assessed for their effects on nerve cell proliferation and regeneration ex vivo and in vivo, with an eye to exploiting this knowledge to define genetic and small molecule approaches to improving the ex vivo growth of neural stem cell precursors and neurones. 4) The phenotype of Nap1l2 deletion will be extensively studied in vivo in the adult mouse and during late embryogenesis using inducible Cre/loxP systems.