Figure
1: Deletion of the Nap1l2 gene leads to neural tube defects in
day 12.5 mouse embryos (arrows).
Nucleosome assembly protein 1 like 2
The murine gene Nap1l2 belongs to a family of genes which encode nucleosome assembly proteins (NAP). These proteins are cellular histone transporters, that are involved in chromatin modification and cell cycle regulation. Nap1l2 is expressed specifically in neuronal cells (see figure 1) of the entire nervous system. Deletion of the gene results in embryonic lethality in the mouse. The embryos exhibit severe neural tube defects similar to anencephaly or spina bifida in human. This knock-out phenotype is due to an overproliferation of neural stem cells, whilst overexpression of Nap1l2 leads to cell cycle arrest and apoptosis.
Figure
1: Deletion of the Nap1l2 gene leads to neural tube defects in
day 12.5 mouse embryos (arrows).
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Our current studies on Nap1l2 are aimed at understanding its role in neurogenesis during normal mouse development and its interaction with other epigenetic mechanisms in the control of the programming of neural stem cells.
The laboratory is using different strategies to study the Nap1l2 function:
An in-vivo model: Conditional knock-out mice carrying a floxed Nap1l2 gene are crossed with transgenic mice expressing Cre recombinase under control of neural specific genes. This allows to study the function of Nap1l2 in the nervous system and its possible role in tumorigenesis.
An ex-vivo model: Conditional knock-out ES cells are differentiated into neurons (figure 2). A genetic selection protocol is used to obtain pure neural precursors. These precursors are then differentiated into post-mitotic neurons. Different experiments comparing deleted and non-deleted cells are undertaken to determine the role of Nap1l2 in neural cell cycle and differentiation.
We aim to identify Nap1l2 target genes by using microarrays and ChIP seq; and Nap1l2 interacting proteins by co-immunoprecipitation and yeast double hybrid approaches.
Deletion of Nap1l2 leads to deficiencies in differentiation and increased maintenance of the neural stem cell stage. Nap1l2 associates with chromatin and interacts with histones H3, H4. Loss of Nap1l2 results in decreased histone acetylation activity leading to 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 and an increase in acetylated histone H3K9/14 at the site of Cdkn1c transcription. These results lead us to suggest that the Nap1l2 protein plays an important role in regulating transcription in developing neurons via the control of histone acetylation.
ES cells growing on gelatine coated dishes
Immunofluorescence of differentiated neurons expressing Tubulin ß III
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. In this context we 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. By two-hybrid analysis we have identified several proteins interacting with NAP1L2, including the ubiquitously expressed members of the nucleosome assembly protein family NAP1L1 and NAP1L4. Structural studies further predict that all five NAP1-like proteins are able to interact directly via their highly conserved alpha-helices. These elements taken in conjunction with the coexpression of all the NAP1-like proteins in neurons and the finding that deletion of Nap1l2 affects both cytoplasmic-nuclear distribution patterns of NAP1L1 and NAP1L4 and their recruitment to target genes, suggests that combinatorial variation within the NAP family may ensure adaptation to the specific requirements for neuronal differentiation such as intercellular repartition, chromatin modification, transcriptional regulation, or the recruitment of specific transcription factors.
In house participants : Mikaël Attia (Postdoc ANR 2011), Philip Avner (Unit head), Magalie Lubineau (M2 in 2009), Christophe Rachez (staff scientist in the Unité de Régulation épigénétique) (protein interactions), Ute Christine Rogner (senior staff scientist, coordinator), Jean Yves Coppée et al. (Plate-Forme 2 - Puces à ADN), Abdelkader Namane et al. (Plateforme 3 - Protéomique)
Outside participants : Andreas Förster & Paul Fremont, UK; Jérome Garin, Grenoble, FR; Geneviève Almouzni & Jean-Pierre Quivy, Paris, FR; Matthieu Gérard et al. CEA, Evry FR; Plateforme Biopuces et Séquençage IGBMC, Illkirch, FR, Hybrigenics Paris, FR
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