Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells

Abstract

Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5 dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)-EGC, and 36 (11.5 dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N-terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N-methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.

PRMT7 Induces Epithelial-to-Mesenchymal Transition and Promotes Metastasis in Breast Cancer

“…5C). Recently, the EMT program has been found to generate breast cancer stem cell (CSC)-like cells (49), and PRMT7 is associated with the pluripotency of mouse embryonic stem cells (50). Although CSCs and embryonic stem cells share some similarities, we did not identify CD44 high /CD24 low cell populations in PRMT7-expressing MCF10A cells by FACS (data not shown), suggesting that the PRMT7-mediated EMT is irrelevant to CSCs in nontumorigenic breast epithelial cells.…”

The Relationship Between Pluripotency and Mitochondrial DNA Proliferation During Early Embryo Development and Embryonic Stem Cell Differentiation

“…Expression of these genes is restricted to the pluripotent cells of pre-implantation embryos, undifferentiated ESCs and primordial germ cells (PGCs) [113][114][115]. Interestingly, overexpression of Nanog and Pramel7 maintains self-renewal and pluripotency in the absence of LIF/ STAT3 stimulation [106,115] whereas overexpression of Oct4 induces differentiation of mESCs into extraembryonic primitive endoderm and mesoderm suggesting that precise levels of Oct4 expression are required for maintenance of self-renewal and pluripotency [108].…”

Large Scale Phosphoproteome Profiles Comprehensive Features of Mouse Embryonic Stem Cells

“…The two different profiles may imply a unique regulatory mechanism for the phosphorylation events of NANOG. Moreover, such modifications of "master" regulators could play a role in the mechanism of pluripotency (62). However, further efforts are needed to study the biological importance and functional significance of this mechanism.…”