Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation

Abstract

Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

Keywords: Ascl1; H2A.Z acetylation; H3K4me3; Tip60/Kat5; bivalent chromatin; cell fate; gene activation; neurogenesis; reprogramming; transcription.

Figure 1.. Tip60 interacts with neuronal lineage-determining factors.

(A) Design of the study. (B) GO enrichment and network analysis of Brn2, Ascl1 and Myt1l interactome. Overrepresented GO Biological Process categories (q-value ≤ 0.05) were clustered into modules and interconnected based on the kappa score (>0.4). Size shows significance, color marks interrelated terms, edge thickness reflects kappa score. (C) Subset of PPI network showing interactions of TFs with acetyltransferase complexes. Colored nodes and edges denote candidates identified in this study, translucent nodes and gray edges represent known PPIs (BioGRID). (D) MYST acetyltransferases identified in this study. Chromo, chromodomain; C2HC-ZF, C2HC-type zinc finger; Ser-rich, serine-rich; CCHHC-ZF, CCHHC-type zinc finger. (E) Fraction of TauGFP⁺ cells at day 5 of reprogramming of TauGFP MEFs transduced with Ascl1 and either vector control, non-targeting control shRNA (shNTC), or shRNAs targeting Tip60, Hbo1 or Mof (n=3). (F) qRT-PCR measuring Tip60, Hbo1 and Mof transcript levels two days post-Ascl1 induction (n=3; error bars, s.d.). (G) TauGFP levels at day 5 of Ascl1 induction (n=3). Statistical significance, (E, G): one-way ANOVA; ****p<10⁻⁴; ***p<10⁻³; ns, not significant, (p>0.05); error bars, s.d.

Figure 2.. Tip60 is a gate keeper of neuronal cell identity.

(A and B) Immunoblotting at day 2 (A) or at indicated times (B) of Ascl1 induction in MEFs. (C) Representative immunostaining of cells from (A) at day 14. (D-G) Absolute (D) or normalized (E-G) TauGFP level at day 5 of reprogramming induced by the combination of the indicated TFs. (H) Representative immunostaining of cells from (D-G) at day 14. (I) Strategy used to test the effect of Cas9/sgRNA-mediated depletion of Tip60 on NPCs differentiation. (J) Immunoblotting at days 0 and 7 of the differentiation of NPCs expressing sgRNA control (Renilla) or Tip60 sgRNAs (sgRNA#3-#5). (K and L) Differentiation of NPCs assessed at day 7 by anti-TUJ1 immunostaining. Shown are (K) representative immunostaining and (L) differentiation efficiency as a fraction of TUJ⁺ cells (n=3). (M and N) NPCs were co-infected with sgRNAs and a vector control or HA-Tip60^WT. Shown are (M) representative immunostaining and (N) differentiation efficiency determined as in (L) (n=3). Statistical significance, (D-G, L, N): one way ANOVA; ****p<10⁻⁴; ***p<10⁻³; ns, not significant (p>0.05); error bars, s.d. Scale bars: 100μm.

Figure 3.. Tip60 acetylates H2A.Z during neuronal induction.

(A) Effect of point and Tip60 truncation mutant proteins on the induction of TauGFP by Ascl1. (Left) Constructs used in structure-function analysis. Point mutations are highlighted in red. PLIP, a splice variant of Tip60. (Right) Fraction of TauGFP⁺ cells at day 5 of Ascl1 induction in presence of shNTC (black) or shTip60#2 (purple), and either vector control or indicated Tip60 constructs. (B) Representative immunostaining of selected conditions from (A) at day 14 (see also Figure S4C). Scale bar: 50μm. (C-F) Immunoblotting of total MEF lysates at day 2 post-dox (C and D) or NPCs lysates at day 4 post-infection (F). (E) Quantification of H2A.Zac signals from (C) (left, n=5) and (E) (right, n=4). (G) In vitro acetylation of recombinant H2A.Z using purified GST-Tip60, or GST alone, in the presence or absence of acetyl-CoA. Statistical significance, (A, E): one way ANOVA; ****p<10⁻⁴; **p<10⁻²; *p<0.05; ns, not significant, (p>0.05); error bars, s.d.

Figure 4.. H2A.Z acetylation is critical for neuronal specification.

(A and B) Immunoblotting (A) and the quantified H2A.Zac intensity (B) at indicated times of Ascl1 induction. (C and D) Comparison of immunoblotting signals (C) and normalized H2A.Zac levels (D) in TauGFP⁺ and TauGFP⁻ Ascl1-iN cells. (E) Fraction of TauGFP⁺ cells at day 5 of Ascl1-induced reprogramming in the presence of increasing amounts of control or shH2A.Z (n≥3). (F) Representative immunostaining of cells from (E) at day 14. (G) Schematics of H2A.Z N- and C-terminal amino acid sequences showing acetylation (Ac) and ubiquitination (Ub) sites, and lysines that were mutated to generate acetylation-resistant H2A.Z^KR. (H) FRAP analysis of Halo-tagged H2A.Z^WT, H2A.Z^KR, H2B, or Halo-NLS. Fluorescence intensity recovery was measured at the bleach spot in MEFs labeled with HaloLigand-JF549, at day 2 (n=21 cells, 3 biological replicates; error bars, s.e.m; NLS, nuclear localization signal). (I) Immunoblotting at day 2 of Ascl1 induction. Quantification of endogenous H2A.Z, normalized to HSP90 and to the vector control is shown below. (J and K) H2A.Zac is required for neuronal induction. Fraction of TauGFP⁺ cells at day 5 (n=6) (J) and representative immunostaining of the quantified cells at day 14 (K) (see also Figure S5L). Statistical significance, (D): unpaired two-tailed Student’s t-test; (B, E and J): one way ANOVA; ****p<10⁻⁴; **p<10⁻²; *p<0.05; ns, not significant, (p>0.05); error bars, s.d. Scale bars: 100μm.

Figure 5.. Tip60 is required for suppression of fibroblast cell identity and induction of neuronal fate.

(A) Fuzzy c-means clustering of differentially expressed genes (DEGs) identified by RNA-seq at days 2 and 5 of Ascl1 induction (n=2, biological replicates; 4,215 DEGs, fold change (fc)≥1.5, padj≤0.05). (B) MA plots of pairwise DE analyses between control and shTip60-expressing Ascl1-iN cells at day 2 (left) or day 5 (right). Upregulated (Up) and downregulated (Down) genes are colored in red and blue, respectively. NS; not significant. (C) GO Biological Process term enrichment analysis of genes upregulated (red) or downregulated (blue) upon Tip60 RNAi in Ascl1-iNs. (D) Number of DEGs detected upon Tip60 RNAi in MEFs or Ascl1-iNs. X-axis shows days post-infection (MEF) or post-induction (Ascl1-iN). (E) ISMARA and predicted TF activity in day 5 Ascl1-iNs. Arrows indicate TF target gene expression (relative to shNTC control): upregulated, red; downregulated, blue. Elevated E-box motif activity likely results from a higher Ascl1 expression in Tip60-depleted cells. (F and G) Impaired expression of Rest targets in shTip60 cells. (F) Day 5 DEGs ranked based on log₂fc, with upregulated and downregulated genes labeled in red and blue, respectively. Position of Rest targets is indicated below. (G) Expression of Rest targets in day 5 Ascl1-iNs (Wilcoxon test, unpaired; ****padj<10⁻⁴; **padj<0.01; *padj<0.05). (H) GSEA of MEF signature in day 5 DEGs (Ascl1+shNTC vs Ascl+shTip60#3). NES, normalized enrichment score. (I) Expression of selected fibroblast and neuronal genes (n=2, RNAseq). (J) PCA of ATAC-seq datasets (n=2, biological replicates). (K) Tip60 knockdown interferes with chromatin “switch” associated with iN reprogramming. Average normalized ATAC-seq read count within ±1.5kb of MEF- and iN-specific open chromatin sites that changed accessibility upon Tip60 RNAi by day 5 (6113 sites, fc≥2, padj≤0.05, with respect to shNTC). Heatmap was sorted based on the fc enrichment between day 5 Ascl1+shNTC and rtTA+shNTC. GO term enrichment of the top 1.5k MEF- and iN-specific sites, and selected genes contributing to the terms (right).

Figure 6.. Tip60 promotes H3K4me3 deposition and silent gene activation.

(A) Classification of Tip60 peaks in MEFs (left) and Tip60 peaks that are differentially enriched between MEFs and day 2 Ascl1-iN (day0-day2), or day 2 Ascl1-iN and day 5 TauGFP⁺ Ascl1-iN (day2-day5), (fc≥2, padj≤0.1). Shown are peaks that gain (Up) or lose (Down) enrichment. (B) E-box motif enrichment at sites gaining Tip60 at day 2. (C) Dynamics of Tip60 binding at Ascl1/Tip60 co-occupied sites (top) and at sites gaining Tip60 in the absence of Ascl1 binding (bottom). (D) Normalized (RPGC) H2A.Zac (left) or H2A.Z (right) read counts at Ascl1/Tip60 co-bound sites (Ascl1 peak summit±250bp). (E-G) Tip60 depletion affects induction of silent (‘off’) genes by Ascl1. (E) Day 5 expression of selected Ascl1 targets (blue, left) and their corresponding ‘on’ (active) and ‘off’ (silent) expression category (right) (see also Methods). (F) Expression of all ‘on’/‘off’ Tip60-regulated Ascl1 targets (padj≤0.01). (G) Association between the direction of the expression change determined for the ‘off’ genes identified among the indicated groups of DEGs. Numbers show Odds ratio; p values are in brackets; n.s., not significant. (H) Expression of genes induced (top) or repressed (bottom) during 5 days of neuronal induction. (I) Normalized read count (RPGC) at the ‘on’ (top) and ‘off’ (bottom) Ascl1 target gene enhancers and promoters was determined for Tip60, H2A.Zac and H2A.Z in TauGFP MEFs (Ascl1 peak summit or TSS, ±250bp), and for H3K4me3 (TSS±500bp) in Cre/ΔCre-transduced Tip60 cKO MEFs. (J) log₂ fc in H3K4me3 read count (RPGC, TSS±500bp), with respect to rtTA+ΔCre, was determined for the ‘on’/‘off’ genes during Tip60 cKO MEF reprogramming. The values were plotted for day 5 DEGs (MEF vs day5 Ascl1-iN, fc≥1.5, padj≤0.05) that were significantly affected by Tip60 depletion. Statistical significance: (D, F, H-J) unpaired Wilcoxon test; ****padj<10⁻⁴; ***padj<10⁻³; *padj<0.05; ns, not significant.

Figure 7.. Tip60/H2A.Zac promote activation of bivalent genes critical for neuronal induction.

(A) Chromatin states (left) and chromatin state enrichment within TSS±2kb of Tip60-regulated ‘off’ genes (right) determined by ChromHMM. (B) Normalized H3K27me3 and H3K4me3 (TSS±500bp), and H2A.Zac and H2A.Z (TSS±250bp) read counts were determined for MEF genes with TSS overlapping ChromHMM states. Shown are: H3K27me3:H3K4me3 ratio (left), H2A.Zac and H2A.Z enrichment (middle), and average gene expression (right). (C and D) qRT-PCR measuring Miat levels in control and Ascl1-iN cells at indicated times (C) (n=3; error bars, s.e.m.), or at day 2 of neuronal induction (D) (n=5; error bars s.d.). (E) ChIP-seq (Ascl1, H3K27me3, H3K4me3(ENCODE)), Cut&Tag (Tip60, H2A.Zac, H2A.Z, H3K4me3) and ATAC-seq signal tracks showing chromatin dynamics at Miat gene locus in MEFs (black) and during neuronal reprogramming. ChromHMM chromatin states defined in (A) are depicted below. Promoter-proximal region (blue) and enhancer region (gray) are shown. (F-G) Fraction of TauGFP⁺ cells at day 5 (n=3; error bars, s.d.) (F), and representative immunostaining at day 14 (G) of Ascl1-induced reprogramming. Statistical significance, (B): unpaired Wilcoxon test; (D, F): one-way ANOVA. ****p<10⁻⁴; ***p<10⁻³; **p<10⁻²; *p<0.05; ns, not significant (p>0.05). Scale bar: 100μm.