Chromatin changes in dicer-deficient mouse embryonic stem cells in response to retinoic acid induced differentiation

Abstract

Loss of Dicer, an enzyme critical for microRNA biogenesis, results in lethality due to a block in mouse embryonic stem cell (mES) differentiation. Using ChIP-Seq we found increased H3K9me2 at over 900 CpG islands in the Dicer(-/-)ES epigenome. Gene ontology analysis revealed that promoters of chromatin regulators to be among the most impacted by increased CpG island H3K9me2 in ES (Dicer(-/-)). We therefore, extended the study to include H3K4me3 and H3K27me3 marks for selected genes. We found that the ES (Dicer(-/-)) mutant epigenome was characterized by a shift in the overall balance between transcriptionally favorable (H3K4me3) and unfavorable (H3K27me3) marks at key genes regulating ES cell differentiation. Pluripotency genes Oct4, Sox2 and Nanog were not impacted in relation to patterns of H3K27me3 and H3K4me3 and showed no changes in the rates of transcript down-regulation in response to RA. The most striking changes were observed in regards to genes regulating differentiation and the transition from self-renewal to differentiation. An increase in H3K4me3 at the promoter of Lin28b was associated with the down-regulation of this gene at a lower rate in Dicer(-/-)ES as compared to wild type ES. An increase in H3K27me3 in the promoters of differentiation genes Hoxa1 and Cdx2 in Dicer(-/-)ES cells was coincident with an inability to up-regulate these genes at the same rate as ES upon retinoic acid (RA)-induced differentiation. We found that siRNAs Ezh2 and post-transcriptional silencing of Ezh2 by let-7 g rescued this effect suggesting that Ezh2 up-regulation is in part responsible for increased H3K27me3 and decreased rates of up-regulation of differentiation genes in Dicer(-/-)ES.

Figure 1. Dicer’s effect on H3K9me2 distribution patterns in mES cells.

A) Differential distribution of H3K9me2 in mouse chromosomes. Blue bars represent loci where higher enrichment levels were observed in mES cells while red bars represent loci where enrichment levels were higher in Dicer^-/- ES cells. Specific genomic loci can be found in table S3 . B) Bar graph showing ratio of H3K9me2 occupation in Dicer^-/- ES cells compared to WT ES cells in different genomic elements. C, D and E) Sequence tags from H3K9me2 ChIP-Seq experiment mapped to the UCSC genome browser to show enrichment at Oct4 and Sox2 and Hoxa gene cluster promoter regions of WT ES and Dicer^-/- ES cells. Blue peaks represent WT ES cells and red peaks represent Dicer^-/- ES cells.

Figure 2. Ezh2 transcripts in Dicer^-/- ES cells are overexpressed compared to WT ES cells, while Setd2, G9a and Ash1l levels remain relatively similar in both cell lines.

A, B C and D bar graphs showing expression levels of the histone methyl transferases Setd2, G9a, Ash1l and Ezh2 in WT and Dicer^-/- ES cells upon RA treatment through days 0 (D0) to day 6 (D6). Note that the transcripts of enzymes Setd2 and Ash1l, which promote H3K36me3 and H3K4me3 modifications respectively show comparable results while H3K9me2 promoting G9a is slightly elevated at a statistically insignificant level in Dicer^-/- ES cells and Ezh2 is significantly high in Dicer^-/- ES cells. The symbol* indicates that the results of a given time point were significantly different between the two cell lines at a confidence level of 0.05 when a students t-test was performed.

Figure 3. Enrichment levels of transcriptionally favorable versus transcriptionally unfavorable histone modifications in pluripotency factor gene promoter regions and their mRNA expression in mES cells.

A, B, C and D left ChIP-qPCR and right panels qRT-PCR results of relative mRNA levels in Oct4, Sox 2, Nanog and Ronin in WT and Dicer^-/-ES cells upon RA induced differentiation through days 0 (D0) to 6 (D6). A and B right panels transcripts of Oct4 and Sox2 go down both in mES and Dicer^-/-ES cells upon RA treatment (A and B left panels). Transcriptionally favorable (H3K4me3) and unfavorable (H3K27me3) presence at Oct4 and Sox2 promoters in WT ES and Dicer^-/- ES (C right panel). Expression levels of Nanog go down upon RA induction while transcriptionally favorable H3K4me3 marks gradually go down in both cell lines (C left panel) (D left panel). H3K4me3 and H3K27me3 occupation at the Ronin promoter (D right panel). Transcripts of Ronin go up at a higher rate in Dicer^-/- ES cells compared to WT ES cells. Figure 3A Left panel * indicates that the difference for a given cell line at a given time point was significant when compared to day zero of the same cell line at 0.05 confidence levels when a students t-test was performed. Figure 3D right panel * indicates that the difference at a given time point between the two cell lines were significant at 0.05 confidence levels when a students t-test was performed.

Figure 4. Transcriptionally favorable H3K4me3 modifications in promoter regions increase expression levels of Lin28b in Dicer^-/- ES cells relative to WT ES cells, while H3K27me3 histone modifications disfavor Hoxa1 and Cdx2 expression in Dicer^-/- ES cells relative to WT

ES. AB C and D left panels, ChIP- qPCR results on promoter regions and qRT-PCR results (Right panles) of Lin28b, Gcnf, Hoxa1 and Cdx2 respectively upon RA induction from day 0 (D0) through day 6 (D6). Note higher enrichment levels of H3K4me3 in the promoter regions of Lin28b (A left panel) which affect increased expression levels in Dicer^-/- ES cells (A right panel). As shown in C and D left panels higher occupation of H3K27me3 in Hox a1 and Cdx2 attenuates upregulation of transcripts in Dicer^-/- ES cells compared to WT ES cells (C and D right panels). The symbol * indicates that the difference at a given time point between the two cell lines were significant at 0.05 confidence levels when a students t-test was performed.

Figure 5. Transfecting Dicer^-/- ES cells with let-7g miRNA rescues Hoxa1 and Cdx2 transcript levels during retinoic acid induced differentiation.

A) Hoxa1 and Cdx2 transcript levels increase upon transfecting Dicer^-/- ES cells with miRNA let-7g while Ezh2 transcript levels go down. B) Lin28b transcripts are significantly reduced in Dicer^-/-ES cells upon transfecting with miRNA let-7g. * indicates that the difference at a given time point between Dicer^-/- ES cells and Dicer^-/- ES cells with let-7g were significant at 0.05 confidence levels when a students t-test was performed.

Figure 6. Transfecting Dicer^-/- ES cells with siRNAs specifically targeting Ezh2 rescues Hoxa1 and Cdx2 transcript levels during retinoic acid induced differentiation.

A) Ezh2 transcript levels reduce while Hoxa1 and Cdx2 transcript levels increase upon transfecting Dicer^-/- ES cells with siRNA specific to Ezh2. B) Lin28b transcripts remain significantly unchanged upon transfecting Dicer^-/-ES cells with siRNA specifically targeting Ezh2. * Indicates that the differences at a given time point between Dicer^-/- ES cells and Dicer^-/- ES cells with siEzh2 were significant at 0.05 confidence levels when a students t-test was performed.

Figure 7. Transfecting Dicer^-/- ES cells with let-7g miRNA and siRNA targeting Ezh2 reduces H3K27me3 at Hoxa1 and Cdx2 during retinoic acid induced differentiation.

Cells were treated with let-7g miRNA and siRNA-targeting Ezh2 along with a scrambled negative control as described in the methods section and chromatin immunoprecipitated using an H3K27me3 specific antibody. Thereafter enrichment of H3K27me3 at Hoxa1 and Cdx2 loci were assayed by quantitative real time PCR. * Indicates that the differences at a given time point between Dicer^-/- ES cells and Dicer^-/- ES cells treated with siEzh2, or let-7g mimic were significant at 0.05 confidence levels when a students t-test was performed.

Figure 8. Putative model explaining Dicer mediated regulation of mES cell differentiation.

When ES cells are induced to differentiate by RA treatment let-7g levels increase. Hence let-7g suppresses Ezh2 resulting in reduced H3K27me3 favoring transcription of Hoxa1 and Cdx2 differentiation genes. As let-7g increases during differentiation Lin28b levels are reduced. As a result translational enhancement of pluripotency factors by Lin28b is reduced, this event in turn favors differentiation.