Attenuating iPSC reprogramming stress with dominant-negative BET peptides

Abstract

Generation of induced pluripotent stem cells (iPSCs) is inefficient and stochastic. The underlying causes for these deficiencies are elusive. Here, we showed that the reprogramming factors (OCT4, SOX2, and KLF4, collectively OSK) elicit dramatic reprogramming stress even without the pro-oncogene MYC including massive transcriptional turbulence, massive and random deregulation of stress-response genes, cell cycle impairment, downregulation of mitotic genes, illegitimate reprogramming, and cytotoxicity. The conserved dominant-negative (DN) peptides of the three ubiquitous human bromodomain and extraterminal (BET) proteins enhanced iPSC reprogramming and mitigated all the reprogramming stresses mentioned above. The concept of reprogramming stress developed here affords an alternative avenue to understanding and improving iPSC reprogramming. These DN BET fragments target a similar set of the genes as the BET chemical inhibitors do, indicating a distinct approach to targeting BET proteins.

Keywords: Peptides; Protein; Stem cell plasticity; Stem cells research.

Graphical abstract

Figure 1

Conserved and masked reprogramming activities of human BET proteins independent of the characteristic bromodomains and ET domain (A) Schematic representations for the deletion constructs of BET proteins. ΔBD12 denotes double deletion, i.e., ΔBD1ΔBD2. (B, D, and E) Relative reprogramming activities of BRD3 (B), BRD2 (D), and BRD4S (E) deletion constructs. FC, fold changes (left Y axis); right Y axis is percentage of reprogramming. Student unpaired two-sided t-tests, n = 3 for BRD4; n = 4 for BRD2; n = 6 for BRD3. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (C) Representative images for ALP staining of the reprogramming wells for different BRD3R constructs under the OSK conditions. Additional transgenes are indicated above each panel. The lower row is for BRD3R deletion constructs. (F and G) Human iPSCs generated by BET deletion mutants are pluripotent as shown by PCA (F) and teratoma tests (G). Bars, 200 μm. See also Figures S1–S4, S10, and S11.

Figure 2

BET proteins displayed conserved and masked mitotic activities (A) Number of enriched mitotic GO terms and mitotic genes for the upregulated gene lists upon forced expression of various BET proteins in the OSK reprogramming cells. (B) 31 mitotic GO terms enriched for the upregulated gene list upon overexpression of BRD2ΔBD1ΔET in the reprogramming cells. FE, fold enrichment; GO, gene ontology. Fisher Exact test; FDR, false discovery rate; FDR <0.05. (C) Heatmaps for the 53 mitotic genes commonly upregulated by BETΔBD1ΔET (BRD2, BRD3, and BRD4S). The heatmaps were based on log2 (normalized read counts), scaled across the rows using the Pheatmap package. (D) BET proteins have concealed mitotic activities in reprogramming cells as shown by cell cycle analyses with BRD4S as an example. ANOVA test, n = 3. ∗, p < 0.05. See also Figures S5–S7 and S12, and Tables S6 and S15.

Figure 3

Almost all commonly downregulated genes by BETΔBD1ΔET with enriched GO terms have roles in stress/stimulus responses (A) The full list of significantly enriched GO terms for the 240 genes commonly downregulated by the BETΔBD1ΔET fragments. Red, stress/stimulus-response GO terms; purple, GO terms for which all the genes have explicit stress/stimulus GO terms as well; numbers in brackets before GO terms are number of genes in the GO term that are not among the 138 genes with enriched stress/stimulus-response GO terms. FE, fold enrichment; GO, gene ontology. Fisher Exact test; FDR, false discovery rate; FDR <0.05. (B and C) Venn diagram (B) showing only 10 of the genes with enriched GO terms does not have annotated stimulus/stress-response GO terms, and the name of these 10 genes (C). (D) Other non-stress-response GO terms in A mainly contain stress/stimulus-response genes with related or other roles. (E) 54% of the 138 downregulated stimulus/stress-response genes represent legitimate down reprogramming.See also Figure S6 and Table S5.

Figure 4

The DN BETΔBD1ΔET fragments predominantly suppress transcription of stress/stimulus-response genes of various categories in naive human fibroblasts (A) 47 selected enriched stimulus/stress-response GO terms with the most gene numbers or representative response categories among the significantly enriched GO terms of the 1,336 shared downregulated genes. The stimulant categories are highlighted in red in the GO terms. FE, fold enrichment; GO, gene ontology. Fisher Exact test; FDR, false discovery rate; FDR <0.05. (B) 56.6% (734 genes) of the mapped genes (1,296 genes) have stress/stimulus-response GO terms of a wide range of categories for the commonly downregulated genes in human fibroblasts. (C) Complete list of enriched stimulus/stress-response GO terms showing exclusive chemical response categories for the upregulated genes by the BETΔBD1ΔET fragments in naive human fibroblasts. (D) Skewed upregulation of chemical response genes by the BETΔBD1ΔET fragments in human fibroblasts.

Figure 5

The bromodomain-null DN BET fragments and chemical targeting of bromodomains commonly downregulate stress/stimulus-response genes (A) BET deletion fragments devoid of individual or both bromodomains commonly downregulated 741 genes. (B) The commonly downregulated genes predominantly have functions of stress/stimulus responses. (C) Heatmaps for the 434 stress/stimulus-response genes downregulated commonly by all the 5 BET deletion mutants in human fibroblasts. Heatmaps were based on averaged normalized read counts in the log2 scale. GFP, n = 3; all mutant RNA-seq, n = 2 except for BRD3RΔBD12, for which n = 3. (D) Complete list of enriched GO terms for the 141 genes commonly downregulated genes in human reprogramming cells by BET chemical inhibitors and BET DN fragments.

Figure 6

The BETΔBD1ΔET fragments mitigate reprogramming stresses (A) Percentage of deregulated gene numbers relative to total expressed genes in the starting cells by overexpression of Yamanaka factors. The epigenetic readers BRD2, BRD3, and BRD4S are used references. The results are based on RNA-seq data. Differentially expressed genes are defined at q < 0.05 and 1.5-fold change levels. (B) Waterfall plots demonstrating the total amount of transcriptional changes by Yamanaka reprogramming factors as measured by cumulative log2(fold changes). (C) 138 stress-response genes are overactivated by OSK but mitigated by the BETΔBD1ΔET mutants in the reprogramming cells. Boxplots in a, b, and c are based on averaged normalized RNA-seq read counts in log2 scale. BJ, n = 8; ESC, n = 7; GFP, n = 3; OSK, n = 3; all BET mutant RNA-seq, n = 2. (D) 65 cell cycle genes are compromised by the reprogramming factors but mitigated by BETΔBD1ΔET fragment in the reprogramming cells. (E) A group of 481 genes were wrongly up-reprogrammed and mitigated by the BETΔBD1ΔET fragments albeit to a lesser extent. (F) Cell cycle profiling by flow cytometry showing compromised cell cycle profiles by OSK reprogramming factors, but mitigated by various BET deletion fragments in the early reprogramming cells. Unpaired, two-sided T test, n = 3. ∗, p < 0.05. (G) Representative histogram of cell cycle profiles determined by flow cytometry, showing compromised G2/M population but attenuated by the BET deletion mutants in the OSK reprogramming cells. (H) Reprogramming factors results in cytotoxicity but mitigated by BETΔBD1ΔET in the reprogramming cells. The same number of cells was seeded at 0 h from cells at 24 h post transduction. The means ± SEM were plotted. n = 3. (I) Summary for attenuation of reprogramming stresses by the dominant-negative BETΔBD1ΔET fragments. See also Figure S9, and Tables S6, S10, S11, S13, and S14.