Poster Presentation Epigenetics 2013

Targeting a multi-copy histone variant gene for knockout using TALEN technology in mice. (#208)

Nur D Anuar 1 , David J Tremethick 1 , Philip D Gregory 2 , Lei Zhang 2 , Josephine Bowles 3 , Tara-Lynne Davidson 3 , Peter Koopman 3 , Ruth M Arkell 4 , Tanya A Soboleva 1
  1. The John Curtin School of Medical Research, The Australian National University, Acton, ACT, Australia
  2. Sangamo Bioscience Inc, Richmond, California, USA
  3. Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
  4. Research School of Biology, The Australian National University, Acton, ACT, Australia

Our work has shown that the exchange of H2A with its variant forms represents one of the most dramatic and essential alterations to the structure and function of chromatin by being required for early metazoan development, chromosome organisation and inheritance, as well as regulating promoter chromatin architecture during the cell cycle and differentiation to determine cell fate. Recently, we discovered a new mouse histone variant, which we designated H2A.Lap1 (Lack of an acidic patch), which, in the adult, is uniquely expressed in the testis and the brain. In the testis, we made the remarkable discovery that H2A.Lap1 is involved in a new mechanism of gene regulation whereby H2A.Lap1 co-ordinately activates the expression of many genes by being targeted to and directly opening the chromatin region encompassing their transcription start site (TSS). However, the role of H2A.Lap1 in the mouse brain remains unknown. An immunohistochemical staining of the brain tissues showed that H2A.Lap1 is expressed in the adult hippocampus and localized in the Purkinje neurons. Interestingly, in undifferentiated mouse neuroblastoma (N2a) cell model, we found that H2A.Lap1 expression was exclusively in cytoplasm and shifted into nucleus upon cells’ differentiation. These observations indicate an important role of H2A.Lap1 in the neurobiology of the central nervous system (CNS). To further understand H2A.Lap1, we are using mouse model to target all three H2A.Lap1 encoded genes located at the X-chromosome. Targeting all three paralogs of H2A.Lap1 (one major and two minor forms) plus a pseudo gene that are 92% identical and located 3.5 Mb from each other using conventional gene targeting is highly challenging. Here, we report the generation of mice with the genetic knock-out of H2A.Lap1. The whole gene family disruption was achieved by employing the Transcription Activator-Like Effector (TALE) Nucleases (TALENs) that mutated all three genes of the H2A.Lap1 family. To our knowledge, this is the first report on TALEN induced mutations on 3 genes simultaneously in mice or any other organism.