Proviral Inactivation of the Npat Gene of Mpv 20 Mice Results in Early Embryonic Arrest

Abstract

The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.The insertion of retroviral proviruses into the host genome can have mutagenic consequences, either by the activation of host genes through juxtaposition of positive viral regulatory elements (7) or by the transcriptional silencing of adjacent host sequences that occurs as murine type C viruses infect germ line cells (15,16). The former phenomenon has been exploited with great success for the discovery of oncogenes; the latter has been useful for identifying mammalian developmental mutations (8, 21). Through retroviral insertional mutagenesis screens, genes whose inactivation caused recessive lethal defects manifest at the midgestation (25), neonatal (22), and adult stages (29) have been identified. The use of defective viruses incorporating selectable markers has further enhanced the utility of the strategy by allowing the infection and selection of ES cell clones in which genes have been trapped (3, 5). There are two great advantages in using retroviruses as mutagenic agents: they generate discrete, localized insertion mutations (as opposed to the large deletions that frequently accompany the insertion of DNA introduced by pronuclear microinjection), and the provirus provides the probe with which the site of insertion can be cloned.We have used the defective murine type C virus myeloproliferative sarcoma virus (MPSV)-neo (23) to infect early mouse embryos in a screen which previously gave rise to the adult kidney-specific mutant strain Mpv 17 (29). In this paper, we describe Mpv 20, a mouse strain in which insertional mutagenesis has resulted in a recessive lethal phenotype manifest at a very early stage of embryonic development. We also report the identification of the mutated gene in Mpv 20 as Npat, a recently described gene closely linked to the Atm locus (13). No function has been reported for Npat, and its predicted amino acid sequence is not sufficiently similar to known sequences to allow predictions about the function of the Npat protein. The data presented herein suggest that whatever its function, the Npat gene protein is absolutely required for development beyond the eight-cell stage. MATERIALS AND METHODS Generation of transgenic mouse lines.Retroviral infection of 4-to 16-cell CFW mouse embryos was accomplished as described previously (15, 29). Briefly, Rat 1 cell...

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription

“…This is supported by the finding that histone mRNA levels are reduced in FLASH À/À morulas, and is consistent with our in vitro data showing that silencing of FLASH results in reduced histone levels and consequent cell-cycle arrest in S-phase (Barcaroli et al, 2006a;Bongiorno-Borbone et al, 2009). These data are also in line with other reports showing that knockdown of other HLB components results in early embryonic lethality (Di Fruscio et al, 1997;Xie et al, 2009). …”

“…Cell-cycle arrest is a direct consequence of reduced histone levels as sustaining histone synthesis through overexpression of Stem Loop Binding Protein (SLBP) prevents the block (Bongiorno-Borbone et al, 2009). Finally, the importance of HLB components for normal progression through the cell cycle is underlined by the early embryonic lethality of homozygous disruption of Hinf-P (Xie et al, 2009) and NPAT (Di Fruscio et al, 1997).…”

Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry

“…Therefore, it appears likely that NPAT is one of the critical targets of cyclin E-CDK2 in promoting S-phase entry. This notion is consistent with the observation that inactivation of the mouse NPAT/NPAT gene by proviral insertion resulted in embryonic arrest at the eight-cell stage (Fruscio et al 1997). …”

The histone gene activator HINFP is a nonredundant cyclin E/CDK2 effector during early embryonic cell cycles

“…Thus, both p220 NPAT and HINFP have a shared molecular function in the activation of histone H4 gene expression (12). The early embryonic lethality of a nontargeted null mutation in p220 NPAT (43) and the genetic demonstration that a defined Hinfp-null mutation also causes early embryonic lethality are consistent with the concept that these proteins are functionally and biochemically linked. The findings that these 2 specific downstream effectors of CDK2/cyclin E kinase are genetically indispensible, whereas CDK2 kinase activity is not, suggest that CDK2 effectors may be more suitable targets for molecular strategies to selectively treat proliferative diseases that include cancer.…”