RESUMO
Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining the genome stability during the cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 leads to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by qRT-PCR profiling and in situ hybridisation [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is multi-interacting protein affecting genome stability on the level of cell division at the earliest stages of development and also by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an additional role in zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp.
Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/fisiologia , Fuso Acromático/metabolismo , Animais , Sistemas CRISPR-Cas , Divisão Celular/genética , Divisão Celular/fisiologia , Desenvolvimento Embrionário/genética , Genes Letais , Instabilidade Genômica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Fosfoproteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Zigoto/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Gastrulation initiates with the formation of the primitive streak, during which, cells of the epiblast delaminate to form the mesoderm and definitive endoderm. At this stage, the pluripotent cell population of the epiblast undergoes very rapid proliferation and extensive epigenetic programming. Here we show that Fam208a, a new epigenetic modifier, is essential for early post-implantation development. We show that Fam208a mutation leads to impaired primitive streak elongation and delayed epithelial-to-mesenchymal transition. Fam208a mutant epiblasts had increased expression of p53 pathway genes as well as several pluripotency-associated long non-coding RNAs. Fam208a mutants exhibited an increase in p53-driven apoptosis and complete removal of p53 could partially rescue their gastrulation block. This data demonstrates a new in vivo function of Fam208a in maintaining epiblast fitness, establishing it as an important factor at the onset of gastrulation when cells are exiting pluripotency.
