Elucidation of the molecular mechanisms of Fam40b for the differentiation of embryonic stem cells

• Applicant: Professor Dr. Agapios Sachinidis, Ph.D.
• Subject Area: Anatomy and Physiology ; Evolutionary Cell and Developmental Biology (Zoology)
• Term: from 2012 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 226334027

We demonstrated that Fam40b is a key regulator of ES cell differentiation towards somatic cells by using an embryonic stem cell line with a constitutive shRNA knockdown of Fam40b (Fam40b KD ES cells or KD ES cells). In contrast to ES cells, Fam40b KD ES cells show upregulation of genes and microRNAs associated with pluripotency even after 16 days of culture under differentiation-inducing conditions (4-, 8-, 12-, and 16-days embryoid bodies (EBs)). In contrast to wt EBs, KD EBs were able to form teratomas in mice. Gene expression data from KD cells suggest that Fam40b preferentially targets genes involved in ncRNA processing, epigenetic regulation of gene expression, and nuclear genes (including nuclear pore genes). Fam40b is mainly located in the nucleus of undifferentiated ES cells and also in the perinuclear region. With increasing differentiation of the ES cells, Fam40b is preferably located in nuclear bodies and becomes tyrosine-phosphorylated. In conclusion, findings from our first DFG grant suggest that our KD Fam40 ES cell model is optimal for studying molecular mechanisms involved in the differentiation of stem cells towards somatic cells. In the present renewal project we aim to 1) to test whether the differentiation-resistant KD ES cell phenotype can be rescued by overexpressing Fam40b (resistant to shRNA) and by introducing miR mimetics and inhibitors regulated by Fam40b allowing cells to differentiate 2) to elucidate molecular differentiation mechanisms of Fam40b by identifying a) DNA targets of Fam40b by ChIP-Seq and b) by identifying Fam40b interaction partners by SILAC proteomics. We also intend to transdifferentiate murine fibroblasts to other cell types by modulating Fam40b-regulated miRs. Finally, the role of the tyrosine phosphorylated Fam40b in the Nerve Growth Factor (NGF)-induced differentiation of PC-12 cells to neurons via tyrosine phosphorylated cascades will be investigated. Overall, the project will significantly contribute to the identification of novel molecular mechanisms of differentiation processes.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Hartmut Schlüter