Histone H4 acetylation and the epigenetic reader Brd4 are critical regulators of pluripotency in embryonic stem cells

Abstract

Background: Pluripotent cells can be differentiated into many different cell types in vitro. Successful differentiation is guided in large part by epigenetic reprogramming and regulation of critical gene expression patterns. Recent genome-wide studies have identified the distribution of different histone-post-translational modifications (PTMs) in various conditions and during cellular differentiation. However, our understanding of the abundance of histone PTMs and their regulatory mechanisms still remain unknown.

Results: Here, we present a quantitative and comprehensive study of the abundance levels of histone PTMs during the differentiation of mouse embryonic stem cells (ESCs) using mass spectrometry (MS). We observed dynamic changes of histone PTMs including increased H3K9 methylation levels in agreement with previously reported results. More importantly, we found a global decrease of multiply acetylated histone H4 peptides. Brd4 targets acetylated H4 with a strong affinity to multiply modified H4 acetylation sites. We observed that the protein levels of Brd4 decreased upon differentiation together with global histone H4 acetylation. Inhibition of Brd4:histone H4 interaction by the BET domain inhibitor (+)-JQ1 in ESCs results in enhanced differentiation to the endodermal lineage, by disrupting the protein abundance dynamics. Genome-wide ChIP-seq mapping showed that Brd4 and H4 acetylation are co-occupied in the genome, upstream of core pluripotency genes such as Oct4 and Nanog in ESCs and lineage-specific genes in embryoid bodies (EBs).

Conclusions: Together, our data demonstrate the fundamental role of Brd4 in monitoring cell differentiation through its interaction with acetylated histone marks and disruption of Brd4 may cause aberrant differentiation.

Fig. 1

Cell morphology and dynamic changes in abundance of histone PTMs. a Retinoic acid differentiation of ESCs results in neuronal cells. From top to bottom, CCE ESCs were grown in suspension for 4 days in the absence of LIF/2i to form embryoid bodies. The embryoid bodies were treated with 1 μM all-trans retinoic acid for 4 days. Cells were plated on gelatin-coated plates and grown for 4 days and 7 days. Red scale bar represents 100 μm. b Relative abundance of histone PTMs during cell differentiation. Heatmap of all single histone modifications from histone H3 and H4, calculated by summing the relative abundance of all peptides carrying each given PTM. A and B represent the two biological replicates for each analyzed time point. The represented value was obtained by averaging the three technical replicates and performing z-score normalization for the rows

Fig. 2

Histone peptide regulation during differentiation. Heatmap of all quantified peptides using nLC-MS from histone H3 and H4. A and B represent the two biological replicates for each analyzed time point. The represented value was obtained by averaging the three technical replicates and performing z-score normalization for the rows.

Fig. 3

Regulation of histone acetylation and Brd4. a Analysis of the acetylated state of the peptide of histone H4 aa 4-17. The singly acetylated form increases during differentiation, while the hyperacetylated states decrease. Above the bars in red the ANOVA p-value (one tail test) among the different conditions is displayed. b Western blotting of Brd4, Oct4 βIII-tubulin and Lamin, this last one used as control. Brd4 decreases during differentiation, and so Oct4. On the bottom, antibody recognizing acetylated histone H4. c qRT-PCR of Oct4 (Pou5f1, in black), Brd2 (in red) and Brd4 (in green) across the five time points of the differentiation process. Actin was used as the reference gene

Fig. 4

Analysis of key regulatory proteins and histone PTMs during JQ1 cell treatment. a (+)-JQ1 treatment of ESCs results in a decrease in Nanog-GFP as measured by FACS. b In triplicate experiments, ESCs were initiated into differentiation in medium lacking LIF. Matched cultures comprising DMSO control (-RA-JQ1), RA treatment for 6 days (+RA-JQ1), JQ1 treatment for 6 days (-RA + JQ1) and RA for 3 days followed by JQ1 for 3 days were maintained and for 6 days with media changes on alternate days. On harvest, mRNA abundance was analyzed by qPCR using a panel of primers (Additional file 1: Table S1). The DDCt data was represented as relative fold over DMSO control (-RA-JQ1). Expression of pluripotency markers, Oct4 and Nanog decreases upon RA and (+)-JQ1 treatment as compared to DMSO control without drug, while expression of germ layer markers increased. The trends of differentiation by inhibition of BET domain by these two treatments and a combination thereof are shown as averages (bottom cells) of multiple markers, respectively for endoderm and neurectoderm. c Most significant histone PTM changes during inhibitor treatment. d Representation of all histone PTMs by plotting their log2 fold change between untreated cells and treated with 100 nM (left) or 200 nM (right) of JQ1 inhibitor. Significant changes were considered when the–test p-value (homoscedastic, two tails) was <0.05, equivalent to >4.32 when transformed into–log2. Color code represents different histone modification types. e Representation of all histone peptide regulations in cells untreated vs treated with JQ1. The two different doses were marked with black and red dots. The figure displays a tight correlation of histone peptide regulation between the two treatments

Fig. 5

Redistribution of Brd4 binding sites during differentiation. a ChIP occupancy for ESC (red) and EB (green) specific Brd4 peaks. Read per million (RPM) was calculated for the Brd4 occupancy in ESC and EBs (b) GO for EB specific peaks. Terms related with neuronal differentiation are enriched (c) GO analysis for the ESC specific peaks

Fig. 6

Genes associated with H4ac and Brd4 binding. Brd4 and H4ac occupancies in ESCs and EBs are shown. a) Constitutive Brd4 peaks near Pou5f1 (b) EB specific increase in Brd4 occupancy at Leafy1 promoter (c) EB-specific Brd4 peaks at Wnt3 promoter (d) ESC specific Brd4 peaks at Il17f promoter