Combined Proteomic and Phosphoproteomic Characterization of the Molecular Regulators and Functional Modules During Pancreatic Progenitor Cell Development.

Differentiated multipotent pancreatic progenitors have major advantages for both modeling pancreas development and preventing or treating diabetes. Despite significant advancements in inducing the differentiation of human pluripotent stem cells into insulin-producing cells, the complete mechanism governing proliferation and differentiation remains poorly understood. This study used large-scale mass spectrometry to characterize molecular processes at various stages of human embryonic stem cell (hESC) differentiation toward pancreatic progenitors. hESCs were induced into pancreatic progenitor cells in a five-stage differentiation protocol. A high-performance liquid chromatography-mass spectrometry platform was used to undertake comprehensive proteome and phosphoproteome profiling of cells at different stages. A series of bioinformatic explorations, including coregulated modules, gene regulatory networks, and phosphosite enrichment analysis, were then conducted. A total of 27,077 unique phosphorylated sites and 8122 proteins were detected, including several cyclin-dependent kinases at the initial stage of cell differentiation. Furthermore, we discovered that ERK1, a member of the MAPK cascade, contributed to proliferation at an early stage. Finally, Western blotting confirmed that the phosphosites from SIRT1 and CHEK1 could inhibit the corresponding substrate abundance in the late stage. Thus, this study extends our understanding of the molecular mechanism during pancreatic cell development.

IFI16 promotes human embryonic stem cell trilineage specification through interaction with p53.

Transcriptional regulation plays an essential role in the self-renewal and differentiation of human embryonic stem cells (hESCs). However, how external signals disrupt the self-renewal regulatory network and further drive hESC differentiation remains largely unknown. Here, we found the immune regulative protein, gamma-interferon-inducible protein 16 (IFI16) was involved in the regulation of both self-renewal and differentiation gene expression during hESC trilineage specification through interaction with p53. IFI16 expression levels were upregulated through JNK activation. IFI16 knockdown delayed the downregulation of self-renewal gene expression and suppressed the upregulation of differentiation gene expression, while IFI16 overexpression accelerated trilineage specification. Furthermore, IFI16 stabilized p53-binding in the genome through IFI16-p53 interaction and differentially regulated self-renewal and differentiation gene expression. Together, our results suggest a particular role of IFI16 in differential gene expression regulation during trilineage specification of hESCs in a manner that is dependent on the genome-wide profile of p53-binding directed by IFI16-p53 interaction.