H3K9 histone acetylation predicts pluripotency and reprogramming capacity of ES cells

Abstract

The pluripotent genome is characterized by unique epigenetic features and a decondensed chromatin conformation. However, the relationship between epigenetic regulation and pluripotency is not altogether clear. Here, using an enhanced MEF/ESC fusion protocol, we compared the reprogramming potency and histone modifications of different embryonic stem cell (ESC) lines (R1, J1, E14, C57BL/6) and found that E14 ESCs are significantly less potent, with significantly reduced H3K9ac levels. Treatment of E14 ESCs with histone deacetylase (HDAC) inhibitors (HDACi) increased H3K9ac levels and restored their reprogramming capacity. Microarray and H3K9ac ChIP-seq analyses, suggested increased extracellular matrix (ECM) activity following HDACi treatment in E14 ESCs. These data suggest that H3K9ac may predict pluripotency and that increasing pluripotency by HDAC inhibition acts through H3K9ac to enhance the activity of target genes involved in ECM production to support pluripotency.

Figure 1

Decreased H3K9ac in E14 ESCs. (A) Immunofluorescence (IF) images of pan-acetylated H4 (H4ac), tri-methylated H3 on lysine 4 and 9 (H3K4me3, H3K9me3), RNA polymerase II phosphorylated on serine 5 (Pol2pS5), HP1α and H3 acetylated on lysine 9 (H3K9ac). (B) Quantitative assessment of (A). Y axis depicts arbitrary fluorescent units. Values represent at least 300 cells from three independent experiments.

Figure 2

HDAC inhibitors increase acetylation and self-renewal. (A) E14 cells were treated with vehicle (DMSO, left) or with TSA for the indicated time, and histone acetylation levels were assayed using antibodies specific for pan-acetylated H3 (H3ac, top) and H3 acetylated on lysine 9 (H3K9ac, bottom). (B) Quantification of (A). (C) Western blots of H3K9ac in E14 ESCs treated with VPA or NaBu for 16 h. Quantification is shown on the right. (D) Increased acetylation of E14 ESC increases teratoma size in vivo. E14 ESC were treated with VPA (0.5 mM) or TSA (5 nM) for 14 h and transplanted under the kidney capsule in SCID mice. Teratomas were analyzed three weeks following transplantation. (E–G) Hematoxylin-Eosin staining of representative sections of teratomas formed by control E14 ESC (E), VPA-treated E14 ESC (F) and TSA-treated E14 ESC (G). All teratomas displayed evidence of endoderm, mesoderm and ectoderm.

Figure 3

VPA increases H3K9ac across the genome. ChIP-seq for H3K9ac in E14 ESCs before (Cont, middle) and after (VPA, top) treatment of VPA (0.5 µM) for 16 h. Unprecipitated DNA was used as input control (Input, bottom). Chromosomal coordinates are shown at the bottom of each gene. Annotated genes above the coordinates denote left-to-right transcripts, annotation below the coordinates denote right-to-left transcripts. (A) H3K9ac profiles of two ECM-related overexpressed genes (Hmcn1, top; Plat, bottom) after VPA treatment. H3K9ac was increased around the transcription start sites of these two genes (gray boxes). (B) H3K9ac profiles for two genes (Atp8a1, top; Ltv1, bottom) which were among the most significantly enriched around the transcription start site for H3K9ac after VPA treatment. (C) H3K9ac profile for an intergenic region on mouse chromosome 17 which displayed a significant increase in H3K9 acetylation.

Figure 4

H3K9ac predicts pluripotency. (A) R1 ESCs, E14 ESCs and MEFs were stained with DAPI (top), Nanog (green) and H3K9ac (red). The levels of Nanog and H3K9ac correlate well. (B) Correlation between Nanog levels (X-axis) and H3K9ac levels (Y-axis).