Die Rolle des Histon-Chaperons Nap1l1 in der Reorganisation des Chromatins während Reprogrammierung und Differenzierung
Allgemeine Genetik und funktionelle Genomforschung
Zusammenfassung der Projektergebnisse
Histone chaperones are proteins that can functionally interact with histones and their variants to assemble/disassemble nucleosomes but are also involved in their nuclear import, storage, or degradation. Histone chaperones contribute to different chromatinrelated processes such as DNA replication, transcription or repair. Dynamic distribution of histone H2A and its variants characterises critical stages of mouse development such as early embryo or germ cell development. The histone chaperone involved in the dynamic distribution of H2A and its variants at these particular stages is not known yet. Nap1l1 is the mammalian homolog of yeast Nap1. Although several studies are describing the role of Nap1 in yeast, plants and flies the impact of Nap1l1 in the dynamic distribution of histones and their variants during mammalian development is still elusive. This research project aims to reveal the unique function of Nap1l1 and how this protein affects chromatin reorganisation during mouse development. To do so, a Nap1l1 knockout murine embryonic stem cell (mESC) line was generated. By using this in vitro system the impact of Nap1l1 on pluripotency, differentiation, gene expression and histone expression/localisation was studied. In addition to this in vitro system, a conditional knockout mouse model was generated to delete Nap1l1 in a tissue specific manner. Nap1l1 depleted mice were used in the context of IVF and natural mating to assess their developmental potential. Surprisingly, Nap1l1 is dispensable for murine germ cell and embryo development. Also in mESCs, Nap1l1 depletion does not affect stem cell maintenance, gene expression or cell proliferation. The mammalian Nap1-like family consist of 5 members. The amino acid sequence of Nap1l4 is very similar to Nap1l1 and so is its expression pattern during mouse development, suggesting that there is functional redundancy between these proteins. To test this hypothesis, Nap1l4 deletion was induced in the conditional Nap1l1f/f background in mESCs and mice. Indeed, Nap1l4 KO mESCs show defects in cell proliferation, gene expression and chromatin compaction. However, loss of both Nap1l1 and Nap1l4 seems not to enhance the phenotype of Nap1l4 KO cells. Despite severe defects on gene expression in Nap1l4 KO mESCs, no developmental defects could be observed in Nap1l4 KO mice. Interestingly, a global knockout of both Nap1l1 and Nap1l4 in mice leads to embryonic lethality. Our results demonstrate for the first time, that both Nap1l1 and Nap1l4 are required for mouse embryo development. These data let us suppose that absence of Nap1l1 or Nap1l4 can be compensated by the other. Additional experiments are required to further characterise the effect of Nap1-like proteins on chromatin compaction in mammalian development and to describe the molecular mechanism behind the observed phenotypes.
