Chromatin-modifying enzymes as modulators of reprogramming

Abstract

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Figure 1. Screening for inhibitor and enhancers of reprogramming

A. Timeline of shRNA infection and iPSC generation. B. Number of Tra-1-60⁺ colonies 21 days after OSKM transduction of 25,000 dH1f cells previously infected with pools of shRNAs against the indicated genes. Representative Tra-1-60-stained reprogramming wells are shown. The dotted lines indicates 3 standard deviations from the mean number of colonies in control wells. C. Validation of primary screen hits that decrease reprogramming efficiency. Fold change in Tra-1-60⁺ iPSC colonies relative to control cells. *P<0.05, **P<0.01 compared to control shRNA-expressing fibroblasts (n = 4; error bars, ± s.e.m). Representative Tra-1-60-stained wells are shown. D. Validation of primary screen hits that increase reprogramming efficiency. Fold change in Tra-1-60⁺ iPSC colonies relative to control cells. *P<0.05, **P<0.01 compared to control shRNA-expressing fibroblasts (n = 4; error bars, ± s.e.m). Representative Tra-1-60-stained wells are shown.

Figure 2. Dot1L inhibition enhances reprogramming efficiency and substitutes for Klf4 and Myc

A. Fold change in the reprogramming efficiency of dH1f cells infected with 2 independent Dot1L shRNAs or co-infected with shRNA-1 and a vector expressing an shRNA-resistant wild-type or catalytically dead mutant Dot1L. Data correspond to the average and s.e.m.; n=independent experiments. *P<0.01 control shRNA-expressing fibroblasts. B. Fold change in the reprogramming efficiency of dH1f cells treated with iDot1L at the indicated concentrations for 21 days. Data correspond to the mean ± s.d.; n=3. *P<0.001 compared to untreated fibroblasts. C. Number of AP⁺ colonies derived from OSKM transduced untreated or iDot1L treated (10um) Oct4-GFP MEFs. *P<0.001 compared untreated MEFs (n = 4; error bars, ± s.d). Representative AP-stained wells are shown. D. Tra-1-60 stained of plates of shCntrl and shDot1L fibroblasts in the absence of each factor or both Klf4 and c-Myc. E. Tra-1-60 stained of plates of untreated and iDot1L treated (3.3uM) fibroblasts in the absence of each factor or both Klf4 and c-Myc. F. Quantification of the Tra-1-60⁺ colonies in Figs. 2d, e representing mean and s.d. of 2 independent experiments done in triplicate.

Figure 3. Nanog and Lin28 are required for enhancement of reprogramming by Dot1L inhibition

A. Overlap of differentially upregulated genes in shDot1L cells 6 Days post-OSKM and OSM transduction with the genes upregulated in OSKM transduced iDotL-treated cells. B. Heat maps showing differential expression levels of commonly upregulated genes in OSKM transduced Dot1L-inhibited cells. C. Number of Tra-1-60⁺ iPSC colonies upon knockdown of Nanog or Lin28 in 2-factor reprogramming of shDot1L cells. Data represent mean and s.e.m of 2 independent experiments done in triplicate. D. Fold-change in Tra-1-60⁺ iPSC colonies in 4-factor (OSKM) and 6-factor (OSKMNL) reprogramming of shCntrl and shDot1L fibroblasts. Data represent mean and s.e.m. of 2 independent experiments done in duplicate. Representative Tra-1-60 stained wells are shown above.

Figure 4. Genome-wide analysis of H3K79me2 marks during reprogramming

A. H3K79me2 ChIP-sequencing tracks (blue) for select EMT-associated genes in fibroblasts and ES cells along with the corresponding H3K27me3 tracks in ES cells (red). B. Expression of EMT-associated transcription factors (EMT-TF) and epithelial genes in control and iDot1L-treated fibroblasts at the indicated time-points during reprogramming. qPCR was normalized to uninfected fibroblasts for EMT-TFs and H1ES cells for Cdh1 and Ocln. C. Number of Tra-1-60⁺ colonies derived from untreated and iDot1L treated (3.3uM) dH1f cells that are either infected with Snail, Twist or Zeb1 expression vectors or treated with soluble TGF-β2 (2ng/ml) (n = 3; error bars, ± s.d.). Representative Tra-1-60-stained wells are shown. D. qRT-PCR quantification of Nanog mRNA level on Day 6 of OSKM expressing untreated or iDot1L-treated (3.3uM) fibroblasts expressing the indicated EMT-factors. Expression levels were normalized to those observed in H1ES cells. E. qRT-PCR quantification of Lin28A mRNA level on Day 6 of OSKM expressing untreated or iDot1L-treated (3.3uM) fibroblasts expressing the indicated EMT-factors. Expression levels were normalized to those observed in H1ES cells.