October 22, 2012;
The roles of the reprogramming factors Oct4, Sox2 and Klf4 in resetting the somatic cell epigenome during induced pluripotent stem cell generation.
ABSTRACT: Somatic cell reprogramming to induced pluripotent stem (iPS
) cells by defined factors is a form of engineered reverse development carried out in vitro. Recent investigation has begun to elucidate the molecular mechanisms whereby these factors function to reset the epigenome.
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Figure 1. The reprogramming assay has revealed enhancer and replacement factors. (a) (i) Example characterization of enhancer factors (X and Y). Factors delivered using individual retroviruses expressing the relevant genes. Nanog serves as a marker of fully reprogrammed cells. Enhancer factors may act through proliferation-dependent (X) or proliferation-independent mechanisms (Y), both of which would increase the proportion of induced pluripotent stem cell colonies. (ii) Example growth curves for mouse embryonic fibroblasts infected with vectors expressing Oct4, Sox2 and Klf4 (O, S and K), and X, Y or control, displaying how proliferation effects can be measured. Error bars represent standard deviation. (b) Example characterization of a Sox2 replacement factor (Z). Error bars represent standard deviation.
Figure 2. Characterization of gene expression changes during MEF reprogramming. (a) Gene expression data were derived from Sridharan et al.  and log2 induced pluripotent stem (iPS) cell/mouse embryonic fibroblast (MEF) expression ratios for all RefSeq genes ordered from highest to lowest. Shown are selected enriched gene ontology (GO) terms for genes with at least a twofold expression difference. (b) (i) Average log2 iPS cell/MEF expression ratios for selected groups of chromatin-modifying enzymes or chromatin-modifying complexes. The red line indicates overall median expression change from (a). (ii-vi) Expression changes for indicated individual complex subunits or specific enzymes between MEFs, pre-iPS cells and iPS cells, normalized to the MEF value. Pre-iPS cells represent embryonic-stem-cell-like colonies that arise during the reprogramming process but do not express pluripotency genes and can be clonally expanded. Expression changes for Taf7 (green), Taf7l (light green), Taf5 (orange), Dpy30 (maroon), Wdr5 (purple), Smarcc1 (BAF155, red) and Smarcc2 (BAF170, blue) are highlighted and discussed in the text. ex., example; Dnmt, DNA methyltransferase; FDR, false discovery rate; TFIID, transcription factor IID; MLL, mixed-lineage leukemia.
Figure 3. A closer look at the reprogramming factors Oct4, Sox2 and Klf4. (a) Important domains of each reprogramming factor, with DNA-binding domains indicated by colored boxes, and transactivation domains underlined in red. HMG, high-mobility group; POU, helix-turn-helix. (b) Reprogramming factor DNA-binding motifs determined by de novo motif discovery. (c) Phylogenetic trees showing the evolutionary relationships between each reprogramming factor and its respective paralogs, based on sequence comparison of their DNA-binding domains. Colors highlight family members that have been tested in the reprogramming assay and are able (green) or unable (red) to mediate reprogramming .
Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo.