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. 2013 Aug;15(4):269-80.
doi: 10.1089/cell.2012.0083. Epub 2013 Jun 15.

Proteomic analysis of early reprogramming events in murine somatic cells incubated with Xenopus laevis oocyte extracts demonstrates network associations with induced pluripotency markers

Alex J Rathbone  1 , Susan Liddell, Keith H S Campbell
Affiliations

Proteomic analysis of early reprogramming events in murine somatic cells incubated with Xenopus laevis oocyte extracts demonstrates network associations with induced pluripotency markers

Alex J Rathbone et al. Cell Reprogram. 2013 Aug.

Abstract

The reprogramming of somatic cells into a pluripotent/embryonic-like state holds great potential for regenerative medicine, bypassing ethical issues associated with embryonic stem cells (ESCs). Numerous methods, including somatic cell nuclear transfer (SCNT), fusion to pluripotent cells, the use of cell extracts, and expression of transcription factors, have been used to reprogram cells into ES-like cells [termed induced pluripotent stem cells (iPSCs)]. This study investigated early events in the nuclei of permeabilized murine somatic cells incubated in cytoplasmic extract prepared from Xenopus laevis germinal vesicle-stage oocytes by identifying proteins that showed significant quantitative changes using proteomic techniques. A total of 69 protein spots from two-dimensional electrophoresis were identified as being significantly altered in expression after treatment, and 38 proteins were identified by tandem mass spectrometry. Network analysis was used to highlight pathway connections and interactions between these identified proteins, which were found to be involved in many functions--primarily nuclear structure and dynamics, transcription, and translation. The pluripotency markers Klf4, c-Myc, Nanog, and POU5F1 were highlighted by the interaction network analysis, as well as other compounds/proteins known to be repressed in pluripotent cells [e.g., protein kinase C (PRKC)] or enhanced during differentiation of ESCs (e.g., retinoic acid). The network analysis also indicated additional proteins and pathways potentially involved in early reprogramming events.

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Figures

FIG. 1.
FIG. 1.
Immunofluorescence labeling analysis of 5-methylcytosine (5MeC) and trimethylated histone H3K9 (H3K9me3) in mouse fibroblasts after no treatment (Control), after permeabilization (Perm control), or after permeabilization and incubation in Xenopus oocyte extract (Extract treated). The intensity of labeling was normalized using the nuclear labeling and the ratios compared for the control, permeabilized control, and Xenopus oocyte extract. Both 5MeC and H3K9me3 labeling was significantly reduced following treatment in Xenopus oocyte extract, when compared to the controls and permeabilized controls. For 5MeC, untreated control 2.30±0.14, permeabilized control 1.40±0.11, treated 0.60±0.07, p<0.0001; analysis of variance (ANOVA) (n=3). For H3K9me3, untreated control 0.92±0.06, permeabilized control 0.82±0.04, treated 0.42±0.03, p<0.0001; ANOVA (n=3).
FIG. 2.
FIG. 2.
Ingenuity Pathways Analysis (IPA) network analysis of proteins identified as affected by Xenopus oocyte extracts in this study. The network is a graphical representation of the molecular relationships in which molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature, a textbook, or canonical information stored in the Ingenuity Pathways Knowledge Base. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation observed from the two-dimensional gel analysis. Various shapes represent different functional classes of the gene product (see key).
See this image and copyright information in PMC

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