Figure 1.
A toy example of gene expression levels during a cellular differentiation process.
(A) Two differentiation events happened at T1 and T2, respectively. From T1, Gene 1 has two expression levels in two subsets of cells in the cell mixture. Gene expression data are available at t0 to t4. (B) The solid black and green lines are not observed after T1 and T2, respectively; instead, the dotted lines are observed as mean expression levels of the cell mixture from microarray data.
Figure 2.
Depletion of the candidate self-renewal factor Smarcad1 by RNAi.
Three shRNA constructs were used to target different regions of respective transcripts. (A) Two days after puromycin selection, the colony morphology of typical undifferentiated ES cells with positive alkaline phosphatase (AP) staining (red) was maintained in two control experiments (Empty and Luci) and three Pias2 knockdown experiments. In contrast, flattened fibroblast-like cells were formed in each Smarcad1 knockdown experiment, and AP staining in Smarcad1 depleted cells was reduced. (B) Quantitative real-time PCR analysis of gene expression in four-day knockdown ES cells. The levels of the transcripts were normalized against the control experiment of empty vector transfection. Data are presented as the mean±SEM, which was derived from three independent experiments.
Figure 3.
A regulatory network in differentiating ES cells.
Modules and regulatory relationships. Yellow and blue nodes represent genes that are up- and down-regulated in differentiated cells. All blue and yellow nodes are collectively termed as pluripotency and differentiation modules, respectively. Edges (plain edges, activators ↑ and repressors ⊤) represent evidence of regulatory relationships. Plain edges: the regulatory relationship is supported by the binding of the regulator to the target gene (ChIP-seq or ChIP-chip data). Activators: the regulatory relationship is supported by both the binding of the regulator to the target gene (ChIP-seq or ChIP-chip data) and down-regulation of the target gene expression when the regulator is knocked down (RNAi microarray data). Repressors: the regulatory relationship is supported by both the binding of the regulator to the target gene (ChIP-seq or ChIP-chip data) and up-regulation of the target gene expression when the regulator is knocked down (RNAi microarray data).
Figure 4.
Enrichment of the RBP-J motif in the upstreams of the differentiation module.
(A) Average upstream binding affinity of RBP-J both shows enhanced signals in the upstream sequences of the differentiation module genes as compared to that of the pluripotency module genes. (B) Testing of all 332 non-redundant mammalian DNA binding motifs available in TRANSFAC v10.2, four motifs were found to be enriched in the upstream sequences of the differentiation module genes as compared to that of the pluripotency module genes (p-value ≤0.05). In particular, the RBP-J motif exhibited the second smallest p-value (0.028) and the largest enrichment factor (2.0) among the 332 motifs.