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

•Tip60 acetyltransferase is essential for neuronal specification•Primary substrate of Tip60 during neuronal induction is histone variant H2A.Z•Tip60/H2A.Zac promote H3K4me3 deposition and activation of bivalent promoters•Bivalent gene activation is necessary for neuronal induction 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. 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. The emergence of new cell fates during development results from dynamic gene expression changes driven by a complex interaction of tissue-specific transcription factors (TFs) with the chromatin and chromatin regulators. 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Here, we set out to identify the key chromatin remodeling complexes that are recruited by proneural TFs to activate chromatin and enable neuronal cell fate specification. We found Tip60 acetyltransferase as critically required for neuronal induction during direct lineage conversion and neuronal differentiation. The mechanism by which Tip60 mediates neuronal specification involves acetylation of histone variant H2A.Z. Remarkably, H2A.Zac is dispensable for overall function of H2A.Z in regulating active transcription and chromatin structure. Instead, it is selectively required for the activation of a subset of silent, lineage-restricted genes marked by bivalent chromatin signature. To identify the key chromatin factors that establish neuronal identity we determined the protein-protein interaction (PPI) network of the three proneuronal TFs, Ascl1, Brn2, and Myt1l, using three experimental approaches (Figures S1A and S1B). 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Invest. 2020; 130: 1431-1445https://doi.org/10.1172/JCI131145Crossref PubMed Scopus (18) Google Scholar As little is known about the role of these proteins in cell fate transition, we decided to explore their function in neuronal induction. To investigate the functional involvement of MYST acetyltransferases in neuronal specification we analyzed how RNAi-mediated depletion of Tip60, Mof, and Hbo1 impacts Ascl1’s ability to induce neuronal identity in TauGFP reporter MEFs (Figures 1E and 1F). 5 days following Ascl1 expression the TauGFP induction in cells co-transduced with Hbo1 and Mof shRNAs was comparable to TauGFP induction in the control cells (Figure 1E). In contrast, knockdown of Tip60 resulted in a striking, ∼80% reduction in TauGFP+ cells on day 5, and absence of MAP2+ neurons on day 14 of neuronal induction (Figures 1E and 2A–2C). RNAi-mediated knockdown of several other subunits of the NuA4/Tip60 complex detected in affinity purification and BioID experiments impaired the induction of TauGFP to a similar degree (Figures 1G and S1D), suggesting a requirement for NuA4/Tip60 complex for neuronal fate specification. Introduction of TIP60 cDNA that is resistant to Tip60 RNAi restored Ascl1-mediated neuronal induction in the knockdown cells (Figures S2A–S2D). To further validate the RNAi effects, we generated a Tip60 conditional knockout (cKO) mouse (Figures S2E–S2H). Indeed, Cre-mediated deletion of Tip60 in cKO fibroblasts impaired the generation of neurons in response to Ascl1 (Figure S2I). Tip60 knockdown did not inhibit lentiviral expression of Ascl1, which instead remained increased throughout the reprogramming (Figure 2B).49Treutlein B. Lee Q.Y. Camp J.G. Mall M. Koh W. Shariati S.A. Sim S. Neff N.F. Skotheim J.M. Wernig M. et al.Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.Nature. 2016; 534: 391-395https://doi.org/10.1038/nature18323Crossref PubMed Scopus (263) Google Scholar Higher efficiency reprogramming systems, such as those that include Brn2 and/or Myt1l in addition to Ascl1, or those that comprise factor combinations without Ascl1,14Chanda S. Ang C.E. Davila J. Pak C. Mall M. Lee Q.Y. Ahlenius H. Jung S.W. Südhof T.C. Wernig M. Generation of induced neuronal cells by the single reprogramming factor ASCL1.Stem Cell Rep. 2014; 3: 282-296https://doi.org/10.1016/j.stemcr.2014.05.020Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar,16Wapinski O.L. Lee Q.Y. Chen A.C. Li R. Corces M.R. Ang C.E. Treutlein B. Xiang C. Baubet V. Suchy F.P. et al.Rapid chromatin switch in the direct reprogramming of fibroblasts to neurons.Cell Rep. 2017; 20: 3236-3247https://doi.org/10.1016/j.celrep.2017.09.011Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar,17Wapinski O.L. Vierbuchen T. Qu K. Lee Q.Y. Chanda S. Fuentes D.R. Giresi P.G. Ng Y.H. Marro S. Neff N.F. et al.Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.Cell. 2013; 155: 621-635https://doi.org/10.1016/j.cell.2013.09.028Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar all failed to override the reprogramming block imposed by Tip60 depletion (Figures 2D–2H). Additionally, knockdown of Tip60 in Ascl1-inducible mouse embryonic stem (mES) cells prevented the formation of neurons also in this system, even though TF-mediated differentiation is extremely efficient and robust in pluripotent stem cells (Figures S2J–S2M).50Zhang Y. Pak C. Han Y. Ahlenius H. Zhang Z. Chanda S. Marro S. Patzke C. Acuna C. Covy J. et al.Rapid single-step induction of functional neurons from human pluripotent stem cells.Neuron. 2013; 78: 785-798https://doi.org/10.1016/j.neuron.2013.05.029Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar Notably, knockdown of Tip60 also impaired the conversion of fibroblasts into muscle-like cells by MyoD1 TF, suggesting that Tip60 function in fate specification is not restricted to neuronal lineage (Figures S2N–S2P). Finally, we found that Tip60 is needed for physiologic neuronal differentiation from embryonic NPCs (Figures 2I–2N). Thus, Tip60 function constitutes a requirement for neuronal fate specification and perhaps for cell fate transition in general. To distinguish at which stage the Tip60 knockdown perturbs neuron formation, we took advantage of an inducible CRISPR-Cas9 system, which relies on the reversible regulation of destabilizing domain (DD)-Cas9 fusion protein by the Shield-1 ligand, to deplete Tip60 at different times during neuronal induction (Figures S3A–S3C).51Senturk S. Shirole N.H. Nowak D.G. Corbo V. Pal D. Vaughan A. Tuveson D.A. Trotman L.C. Kinney J.B. Sordella R. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.Nat. Commun. 2017; 8: 14370https://doi.org/10.1038/ncomms14370Crossref PubMed Scopus (93) Google Scholar We tested the effects of various 3 day-long periods of Shield-1 treatment (Figure S3D). Tip60 depletion during the first 3 days blocked TauGFP induction and generation of TUJ1+ neurons (Figures S3E–S3G). However, as the Shield-1 treatment was progressively delayed, the impact of Tip60 inactivation on neuron formation became less severe, with day 10 treatment having no apparent effects (Figures S3E and S3F). Late and continuous inactivation of Tip60 did not affect neuronal stability when analyzed at day 21 post-induction, i.e., 11 days after Tip60 depletion (Figures S3E and S3F). This suggests that the major role of Tip60 is at the initial steps of the cell identity transition, as opposed to maturation and maintenance of already formed neurons. Given the reported involvement of Tip60 in regulating cell viability and proliferation,26Urban I. Kerimoglu C. Sakib M.S. Wang H. Benito E. Thaller C. Zhou X. Yan J. Fischer A. Eichele G. TIP60/KAT5 is required for neuronal viability in hippocampal CA1.Sci. Rep. 2019; 9: 16173https://doi.org/10.1038/s41598-019-50927-1Crossref PubMed Scopus (8) Google Scholar,52Acharya D. Nera B. Milstone Z.J. Bourke L. Yoon Y. Rivera-Pérez J.A. Trivedi C.M. Fazzio T.G. TIP55, a splice isoform of the KAT5 acetyltransferase, is essential for devel