Asf1a resolves bivalent chromatin domains for the induction of lineage-specific genes during mouse embryonic stem cell differentiation

Abstract

Bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications are barriers for the expression of lineage-specific genes in ES cells and must be resolved for the transcription induction of these genes during differentiation, a process that remains largely unknown. Here, we show that Asf1a, a histone chaperone involved in nucleosome assembly and disassembly, regulates the resolution of bivalent domains and activation of lineage-specific genes during mouse ES cell differentiation. Deletion of Asf1a does not affect the silencing of pluripotent genes, but compromises the expression of lineage-specific genes during ES cell differentiation. Mechanistically, the Asf1a-histone interaction, but not the role of Asf1a in nucleosome assembly, is required for gene transcription. Asf1a is recruited to several bivalent promoters, partially through association with transcription factors, and mediates nucleosome disassembly during differentiation. We suggest that Asf1a-mediated nucleosome disassembly provides a means for resolution of bivalent domain barriers for induction of lineage-specific genes during differentiation.

Keywords: bivalent chromatin domain; embryonic stem cell differentiation; histone chaperone; nucleosome disassembly.

Fig. 1.

Asf1a or Asf1b KO does not affect mouse ES cell growth and identity. (A) Western blot analysis of Asf1a levels (Top) in two independent WT, Asf1a KO (aKO), and Asf1b KO (bKO) ES cell clones (generated by different sgRNAs). Ponceau S staining (Bottom) was used as a loading control. (B) Growth curves of two independent WT, aKO, and bKO ES cell lines. (C) BrdU/propidium iodide FACS analysis of WT, aKO, and bKO ES cells. The percentage of cells at each stage of the cell cycle was quantified. The original FACS profiles are shown in SI Appendix, Fig. S1C. (D) Representative AP staining images of WT, aKO, and bKO ES cells. (Scale bar: 50 µm.) (E) RT-PCR analysis of the expression of three pluripotent genes (Nanog, Oct4, and Sox2) in two independent WT, aKO, and bKO ES cell lines. The y axis indicates the relative mRNA level to GAPDH. The results are from three independent experiments, and bars represent mean ± SEM.

Fig. 2.

Asf1a KO affects expression of lineage-specific genes during ES cell differentiation. (A) Representative images of EB morphology during EB differentiation at days 3, 5, 7, and 10 from WT, aKO, and bKO cells. (Scale bar: 1,000 µm.) (B) The diameter of at least 50 EBs at each time point was measured by using ImageJ. (C and D) RT-PCR analysis of pluripotent (C) and lineage-specific gene (D) expression during ES cell differentiation. Data are from three independent experiments. Error bars represent mean ± SEM. The P value was calculated by using a t test between WT and aKO lines (*P < 0.05). The expression of additional germ-layer genes as well as these genes in another independent clone is shown in SI Appendix, Fig. S3 B and C. Note that the effect of Asf1a deletion on the expression of Gata4, although apparent, was not statistically significant for this set of three repeats. The effect of Asf1a deletion on the expression of Gata4 in the rescue experiments shown in Fig. 5F was statistically significant. The difference between these two sets of experiments likely reflects the fact that Asf1a KO on Gata4 expression is small, and therefore some experimental variations during differentiation can mask the difference.

Fig. 3.

Asf1a KO affects dynamic changes of histone modifications at lineage-specific genes during differentiation. (A and B) Analysis of H3K4me3 (A) and H3K27me3 (B) at the promoters of two pluripotent genes (Nanog and Oct4) by ChIP-PCR. (C and D) Analysis of H3K4me3 (C) and H3K27me3 (D) at the promoters of two lineage-specific genes (Gata4 and Gata6). Black and red bars represent ES cells and day 10 EBs, respectively. Data were from three independent experiments. Error bars represent mean ± SEM (*P < 0.05 and **P < 0.01).

Fig. 4.

Asf1a is required for induction of lineage-specific genes during neural differentiation. (A) An outline of the monolayer ES cell in vitro neural differentiation. (B) Gene-expression analysis of Sox21 and Zfpm2 in Asf1a-KO cells expressing EV (aKO) or WT Asf1a (WT) during neural differentiation using RT-PCR. Data are from three independent experiments. Error bars represent mean ± SEM. The P value was calculated by using a t test between WT and aKO NPs (*P < 0.05). (C) Snapshot of RNA-seq results at the Sox21 locus. Data are from two independent experiments (rep1 and rep2). (D) The hierarchical clustering analysis of the differentially expressed genes during neural differentiation of WT and aKO cells identified by RNA-seq. (E) The percentage of bivalent genes in each of the three subgroups of genes identified in D. (F) GO analysis of the group 1 genes. The top 10 significant GO terms and P value are displayed. (G) Relative levels of H3K4me3 and H3K27me3 between WT and aKO at the promoters of three subgroups of genes identified in D. The y axis represents the log2 ratio of ChIP-seq reads between WT and aKO lines. (H) Analysis of changes in H3K4me3 and H3K27me3 at the gene promoter of Sox21 in WT and aKO lines by ChIP-PCR. The P value was calculated by using a t test between ES cells and NPs (*P < 0.05 and **P < 0.01). Note that Asf1a-KO and WT clones were the same as used in Fig. 5.

Fig. 5.

The Asf1a–histone interaction is important for the induction of gene transcription during differentiation. (A) Analysis of the exogenously expressed Asf1a and mutants in Asf1a-KO ES cells. “WT” indicates FLAG-tagged WT Asf1a; “EDAA” indicates Asf1a (E36A, D37A, indicated as EDAA), and “V94R” indicates Asf1a (V94R). Two independent lines were analyzed by using Western blot and compared with endogenous Asf1a level. (B) Analysis of interactions of Asf1a and Asf1a mutants with histone H3 and chaperones HIRA and CAF-1 (p60). Asf1a-KO cells expressing FLAG-tagged WT or Asf1a mutants were immunoprecipitated with M2 beads, and coimmunoprecipitated proteins were analyzed by Western blot. (C and D) Representative images of day 7 EBs (C) and the average size of EBs formed from ES cells expressing WT Asf1a and Asf1a mutants (D). Two independent rescue lines were analyzed, and the results of another line are shown in SI Appendix, Fig. S5A. (Scale bar: 1,000 µm.) (E and F) RT-PCR analysis of pluripotent (E) and four lineage-specific genes (F) of ES clones expressing WT or mutant Asf1a during differentiation. The expression of additional germ-layer marks and the expression results obtained from another independent line are shown in SI Appendix, Fig. S5 B and C [*P < 0.05, (WT+EV) vs. (aKO+EV); ^#P < 0.05 (WT+EV) vs. (aKO+V94R)].

Fig. 6.

Asf1a binds to the promoters of lineage-specific genes. (A and B) Asf1a does not bind to the promoters of Nanog and Oct4 (A) but binds to the promoters of Gata4 and Gata6 (B). Asf1a ChIP-PCR was performed by using ES cells expressing WT Asf1a and Asf1a mutants described in Fig. 5A. Results are from three different experiments, and error bars represent mean ± SEM (*P < 0.05 and **P < 0.01). (C) Asf1a, but not Asf1b, was coimmunoprecipitated with Nanog and Oct4. (D) Identification of TF binding sites (TFBSs) that are enriched in group 1 but not group 2 genes or vice-versa. Top hits were listed together with the −log2 (P value). (E) Expression of Sox9 in ES cells and NPs during neural differentiation based on RNA-seq reads. (F) Asf1a binds to the promoter of Sox9. FLAG ChIP was performed in aKO ES cells expressing EV or WT FLAG-Asf1a, and ChIP DNA was analyzed by quantitative PCR at the Sox9 promoter (*P < 0.05).

Fig. 7.

Asf1a mediates nucleosome disassembly at lineage-specific gene promoter. (A and B) Nucleosome occupancy at two pluripotent genes (Nanog and Oct4, A) and two lineage-specific genes (Gata4 and Gata6, B) during EB formation. H3 ChIP was performed by using chromatin from Asf1a-KO ES cells and day 7 (D7) EBs expressing EV, WT, or mutant Asf1a. Quantitative PCR was performed targeting each of three nucleosomes upstream of TSSs of two pluripotent genes (A) and two lineage-specific genes (B). (C) Nucleosome occupancy at two bivalent gene (Sox21 and Zfpm2) promoters and two pluripotent gene (Nanog and Oct4) promoters during neural differentiation. The H3 ChIP enrichment was normalized against its enrichment at the gene body of GAPDH, and the nucleosome occupancy of ES cells in each line was set as 1. Results are from three different experiments, and error bars represent mean ± SEM (*P < 0.05 and **P < 0.01). (D) A working model depicting that Asf1a mediates nucleosome disassembly at lineage-specific gene promoters and facilitates subsequent association of TFs.