Chromatin Assembly Factor 1 (CAF-1) facilitates the establishment of facultative heterochromatin during pluripotency exit

Abstract

Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.

Figure 1.

Mouse CAF-1 p150 is dispensable for ESC self-renewal. (A) Sanger sequencing analysis of two different p150 KO clones generated by two different sgRNAs. Sequence alignments show frame-shift mutations in KO clones. (B) WB analysis of p150 proteins in WT and two different p150 KO lines. GAPDH (bottom) was used as loading control. (C) Growth curves of two p150 WT and two p150 KO lines. The results are from three independent experiments and bars represent means ± SEM (**P < 0.01, two-tailed Student's t-test). (D) Bright field images show the morphology of spheres derived from p150 WT and KO clones. Scale bar: 50 μM. (E) Representative confocal images of p150 WT and KO lines seeded on feeder cells and stained for Oct4. Scale bar: 20 μM. (F) WB analysis of Oct4, Sox2 and Nanog expression in p150 WT and KO lines. GAPDH was used as loading control.

Figure 2.

Mouse CAF-1 p150 is required for silencing of pluripotency genes during ESC differentiation. (A) RT-qPCR analysis of expression of three pluripotency genes during EB formation. Results are from three independent experiments. Error bar represents means ± SEM. *P < 0.05, ***P < 0.001 (two-tailed Student's t-tests between p150 WT and each KO line). (B) WB analysis of Oct4 and Nanog during EB formation. Tubulin (bottom) was used as loading control. (C) Hierarchical cluster analysis of differentially expressed genes before (ESC, day 0) and after (EB, day 7) differentiation of p150 WT and KO cells identified by RNA-seq. Results from two independent repeats (rep1 and rep2) are shown. (D) GO analysis of the Group 1 genes identified in C. (E) Representative EGFP fluorescence images of two independent reporter lines of p150 WT and KO ESCs during EB formation. The expression of EGFP is driven by the Oct4 distal enhancer. Scale bar: 400 μM.

Figure 3.

The PIP2 region of p150 is important for silencing of pluripotency genes. (A) RT-qPCR analysis of expression of three pluripotency genes (Oct4, Sox2 and Nanog) in p150 KO cells expressing either full-length p150 or empty vector during EB differentiation. Two independent lines of full-length (FL) or empty vector (Vec) were used for the analysis. Results were from three independent experiments (means ± SEM, *P < 0.05, **P < 0.01, two-tailed Student's t-test between p150 KO and KO + FL lines). (B) Analysis of interaction of p150 and p150 mutants with p60, PCNA and histone H3. EGFP-tagged full-length p150 and p150 mutants with deletions of the indicated regions were expressed in p150 KO cells and immunoprecipitated with antibodies against GFP. Co-purified proteins were analyzed by WB. PIP1Δ, PIP1 domain deletion; PIP2Δ, PIP2 domain deletion; MIRΔ, HP1-interacting domain (MIR) deletion. (C) RT-qPCR analysis of expression of three pluripotency genes (Oct4, Sox2 and Nanog) from p150 KO lines expressing WT p150 and p150 mutants during EB differentiation. Results represent means ± SEM of three independent experiments (*P < 0.05, **P < 0.01, two-tailed Student's t-test between WT and KO + PIP2Δ lines). Results from additional lines are shown in Supplementary Figure S4.

Figure 4.

The PCNA mutant defective in CAF-1 interaction is also defective in silencing of pluripotency genes. (A) A schematic representation of generating PCNA R61A D63A mutations using CRISPR/Cas9-mediated homologous recombination. Red asterisk indicates the PCNA mutation sites. The target region was amplified by PCR and sequenced by Sanger sequencing. F, forward primer. R, reverse primer. PAM, protospacer adjacent motif. (B) Analysis of p150–PCNA interaction in mouse ESCs expressing WT and mutant PCNA. EGFP-tagged p150 was expressed in PCNA WT and mutant cells and immunoprecipitated using antibodies against GFP. Co-immunoprecipitated proteins were analyzed by WB. Hetero, one allele with the R61A D63A mutation and one with WT PCNA; Homo, both alleles mutated to R61A, D63A. (C) RT-qPCR analysis of expression of Oct4, Sox2 and Nanog in PCNA WT and mutant lines during EB differentiation. Homo, PCNA homozygous mutation. Results represent means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, two-tailed Student's t-test between PCNA WT and Homo mutant lines).

Figure 5.

CAF-1–PCNA-mediated nucleosome assembly is important for histone modifications at the promoters of pluripotency genes during ESC differentiation. (A andB) ChIP-qPCR analysis of H3K27me3 (A) and H3K4me3 (B) occupancy of promoter regions of Oct4, Sox2 and Nanog in p150 WT and p150 KO lines during EB differentiation. Both ESC and day 10 EB were used for analysis. (C andD) ChIP-qPCR analysis of H3K27me3 (C) and H3K4me3 (D) levels at promoter regions of Oct4, Sox2 and Nanog at different time points during differentiation in p150 WT and p150 KO lines. Results are means ± SEM (n = 3, *P < 0.05, **P < 0.01, two-tailed Student's t-test). (E andF) ChIP-qPCR analysis of H3K27me3 (E) and H3K4me3 (F) occupancy at promoter regions of Oct4, Sox2 and Nanog in PCNA WT and Homo mutant ESC and day10 EB. Results are from three independent experiments (means ± SEM, n = 3, *P < 0.05, **P < 0.01, ^****P < 0.0001, two-tailed Student's t-test).

Figure 6.

Deletion of p150 impairs chromatin dynamics during ESC differentiation. (A) Representative browser tracks showing H3K27me3 and H3K4me3 at the Oct4 and Nanog loci before (ESC) and after (EB, day 7 EB) differentiation. (B–G) Relative levels of H3K27me3 and H3K4me3 between p150 WT and KO cells at the promoters of gene groups identified in Figure 2C. H3K27me3 for Group 1 (B), Group 2 (D), Group 3 (F) and Group 4 (H); H3K4me3 for Group 1 (C), Group 2 (E), Group 3 (G) and Group 4 (I). The y-axis represents the log2 ratio of ChIP-seq reads. The P-values were calculated using Wilcoxon test.

Figure 7.

Deficiency of p150 disrupts the formation of facultative silent chromatin. (A) DNA fragment distribution after MNase digestion on p150 WT and p150 KO ESC. Chromatin from fixed p150 WT and KO ESC was digested by titrated MNase. After purification, DNA was analyzed by agarose gel electrophoresis and DNA size corresponding to mono-, di- and tri-nucleosome is indicated. Image is representative from three independent experiments. Quantification of the signal intensity over indicated lane is shown on the right by ImageJ/plot profiler. A.U., arbitrary units. (B) Nucleosome occupancy at two pluripotent genes (Oct4 and Nanog), two lineage-specific genes (Gata4 and Gata6) and housekeeping gene (GAPDH). H3 ChIP was performed using chromatin from p150 WT and KO cells. Quantitative PCR was performed targeting the nucleosomes upstream of transcription start sited (TSS) of the indicated genes. Results are from three independent experiments. (means ± SEM, n = 3, *P < 0.05, **P < 0.01, two-tailed Student's t-test) (C) Nucleosome occupancy at two pluripotent genes (Oct4 and Nanog). H3 ChIP was performed using chromatin from ESC and day7 EB of p150 WT and KO cell line. Quantitative PCR was performed targeting the −1 nucleosomes upstream of TSS of the indicated genes. Results are from three independent experiments (means ± SEM, n = 3, *P < 0.05, **P < 0.01, two-tailed Student's t-test). (D and E) CAF-1 interacts with Ezh2. (D) EGFP-tagged full-length p150 expressed in p150 KO cells was immunoprecipitated using antibodies against GFP. Co-purified proteins were analyzed by WB using the indicated antibodies. (E) Ezh2 was immunoprecipitated using antibodies against Ezh2 and extracts prepared from p150 KO cells expressing EGFP-tagged p150. Co-purified proteins were analyzed by WB using the indicated antibodies. Immunoprecipitation using IgG was used as a control. (F and G) Impacts of p150 KO on histone and Ezh2 binding at replicating chromatin. (F) Peptides ratio of indicated proteins between p150 WT and KO detected from SILAC-iPOND-MS. ES cells cultured with light isotope growth media (p150 WT) and heavy isotope growth media (p150 KO) were pulsed with EdU to label nascent chromatin and fixed with formaldehyde. Heavy and light labeled cells were mixed 1:1 before click chemistry reaction. After Click reaction to conjugate EdU with biotin, DNA–protein complex on nascent chromatin was purified and eluting proteins were analyzed by mass spectrometry. (G) Eluted proteins from iPOND procedures were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting for indicated antibodies. Representative blots from three independent iPOND experiments were shown.