Histone deacetylase 1 (HDAC1), but not HDAC2, controls embryonic stem cell differentiation Oliver M. Dovey, Charles T. Foster, and Shaun M. Cowley1 Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom Edited* by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved March 30, 2010 (received for review January 13, 2010) Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that complexes, including Sin3A (20), SDS3 (21), MBD3 (22), and help regulate chromatin structure as the core catalytic components LSD1 (23, 24). Germ-line deletion of HDAC1 results in early of corepressor complexes. Although tissue-specific deletion of embryonic lethality around embryonic day (e)10.5, although ab- HDAC1 and HDAC2 has demonstrated functional redundancy, errant development occurs as early as e7.5. In contrast to these germ-line deletion of HDAC1 in the mouse causes early embryonic early embryonic phenotypes, constitutive HDAC2 knockout mice lethality, whereas HDAC2 does not. To address the unique re- survive embryogenesis and either die shortly after birth in one quirement for HDAC1 in early embryogenesis we have generated model (19) or survive to adulthood in others (25–27), albeit at conditional knockout embryonic stem (ES) cells in which HDAC1 or reduced Mendelian frequencies. In a number of cell types, de- HDAC2 genes can be inactivated. Deletion of HDAC1, but not letion of both HDAC1 and HDAC2 is required to generate HDAC2, causes a significant reduction in the HDAC activity of Sin3A, a phenotype (14, 19, 28). This result suggests that the activity of NuRD, and CoREST corepressor complexes. This reduced corepressor HDAC1 and HDAC2 is mostly redundant, with the requirement activity results in a specific 1.6-fold increase in histone H3 K56 for both HDAC1 and HDAC2 occurring only at certain key de- acetylation (H3K56Ac), thus providing genetic evidence that velopmental periods, such as gastrulation. To investigate the es- H3K56Ac is a substrate of HDAC1. In culture, ES cell proliferation sential role of HDAC1 during early embryogenesis we have was unaffected by loss of either HDAC1 or HDAC2. Rather, we find compared the biochemical, proliferative, and differentiation that loss of HDAC1 affects ES cell differentiation. ES cells lacking properties of HDAC1 and HDAC2 conditional knockout embry- either HDAC1 or HDAC2 were capable of forming embryoid bodies onic stem (ES) cells. ES cells are the in vitro counterpart of epi- (EBs), which stimulates differentiation into the three primary germ blast cells of the early postimplantation embryo and their fi fi layers. However, HDAC1-de cient EBs were signi cantly smaller, differentiation mimics many of the changes in gene expression showed spontaneous rhythmic contraction, and increased expres- associated with embryonic development (29). We find that loss of sion of both cardiomyocyte and neuronal markers. In summary, our HDAC1, but not HDAC2, reduces the level of HDAC activity genetic study of HDAC1 and HDAC2 in ES cells, which mimic the associated with HDAC1/2 complexes and leads to the enhanced fi embryonic epiblast, has identi ed a unique requirement for HDAC1 differentiation of embryoid bodies. in the optimal activity of HDAC1/2 corepressor complexes and cell fate determination during differentiation. Results Generation of Conditional Knockout ES Cell Lines for HDAC1 and corepressor | acetylation | deacetylation | chromatin HDAC2. Beginning with an E14 ES cell line expressing an inducible Cre/Estrogen Receptor (CreER) construct from the endogenous lass I histone deacetylases (HDAC1, -2, -3, and -8) are highly ROSA26 locus (30), we used homologous recombination to Cconserved enzymes present in the nucleus of all cells, where produce HDAC1Lox/Lox; CreER and HDAC2Lox/Lox; CreER cell they help modulate levels of gene expression (1). The best lines in which exon 2 of each gene is flanked by LoxP sites (see characterized substrates of HDACs are the N-terminal tails of Fig. 1A and Figs. S1 and S2 for detailed methods). Induction of the core histones H2A, H2B, H3 and H4. Deacetylation of his- Cre activity by addition of 4-hydroxy tamoxifen (OHT) to the tone tails results in a “tightening” of chromatin, due to the growth media resulted in complete recombination of each allele Δ Δ Δ Δ electrostatic potential of unacetylated lysine residues to promote and deletion of exon 2 (HDAC1 2/ 2 or HDAC2 2/ 2) within 24 h internucleosomal interactions (2, 3) and the loss of a binding site (Fig.1B). Loss of exon 2 disrupts the ORF of both HDAC1 and for components of the transcriptional machinery containing a HDAC2 such that a premature stop codon is introduced in bromodomain, e.g., TAFII1 (4). exon 3. Following deletion of exon 2 a further 4–5 days of culture Among the class I HDACs, HDAC1 and HDAC2 are the most are required before HDAC1 and HDAC2 protein levels are re- similar (83% amino acid identity), sharing an almost identical duced below 10% of those of control cells (Fig. 1C), suggesting catalytic core domain and a conserved C-terminal tail (5). In that a combination of a long-lived mRNA and recruitment into mammalian cells HDAC1 and HDAC2 interact together (6) to a stable protein complex results in relatively slow protein turn- form the catalytic core of a number of higher-order complexes over. In the subsequent experiments, HDAC1Lox/Lox; CreER and including Sin3A, NuRD, CoREST, and NODE (reviewed in ref. HDAC2Lox/Lox; CreER cells 10–24 days postrecombination (hence Δ Δ Δ Δ 1). These complexes are then targeted to chromatin by sequence- referred to as HDAC1 2/ 2 and HDAC2 2/ 2) were used to assess specific (often cell-specific) transcription factors to repress their biochemical and growth properties. transcription in cooperation with other chromatin modifiers, such as the lysine-specific demethylase, LSD1, in the CoREST – complex (7 9). As part of these multiprotein complexes, the Author contributions: O.M.D. and S.M.C. designed research; O.M.D., C.T.F., and S.M.C. activity of HDAC1 and HDAC2 has been implicated in the performed research; O.M.D. contributed new reagents/analytic tools; O.M.D., C.T.F., regulation of cell cycle progression by tumor suppressors (10– and S.M.C. analyzed data; and O.M.D. and S.M.C. wrote the paper. 12), differentiation (13, 14), cellular aging (15), and cancer (16). The authors declare no conflict of interest. Indeed, a variety of HDAC inhibitors that target both HDAC1 *This Direct Submission article had a prearranged editor. and -2 are currently being tested in the clinic as potential anti- Freely available online through the PNAS open access option. cancer therapeutics (17). 1To whom correspondence should be addressed. E-mail: [email protected]. Mouse genetics has demonstrated an essential role in embryo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genesis for HDAC1 (18, 19) and many components of HDAC1/2 1073/pnas.1000478107/-/DCSupplemental. 8242–8247 | PNAS | May 4, 2010 | vol. 107 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1000478107 Downloaded by guest on September 30, 2021 Fig. 1. Generation of HDAC1 and HDAC2 conditional knockout ES cell lines. (A) An E14 ES cell line constitutively expressing a Cre/Estrogen Receptor (CreER) fusion from the ROSA26 locus was used to generate homozygous conditional knockout alleles for HDAC1 and HDAC2. Both copies of exon 2 were flanked by LoxP sites, using consecutive rounds of gene targeting. (B) Southern blots showing wild-type and exon 2 targeted HDAC1Lox/Lox and HDAC2Lox/Lox loci. Addition of 4-hydroxy tamoxifen (OHT) activates the Δ Δ Δ Δ CreER fusion and induces deletion of exon2 (HDAC1 2/ 2, HDAC2 2/ 2)inES cells; >95% recombination is observed after 24 h. (C) Quantitative Western blot shows ligand-inducible deletion of HDAC1 and -2 protein in whole-cell extracts from HDAC1Lox/Lox and HDAC2Lox/Lox ES cells, respectively. Cells were cultured for up to 10 days (0–3 days in the presence of OHT). α-Tubulin was used to normalize protein loading. Blots were visualized and quantitated using a LiCOR scanner. Data are representative of three independently tested clones for each genotype. CELL BIOLOGY Reduced HDAC Activity Associated with HDAC1/2 Complexes in the Absence of HDAC1. Consistent with previous reports (18, 31), deletion of HDAC1 results in an increased level of HDAC2 protein (Fig. 2A). However, no increase in HDAC2 mRNA is detected, suggesting that increased protein levels may occur from changes in HDAC2 translation and/or degradation in the ab- sence of HDAC1. Interestingly, there is no change in HDAC1 protein levels in the absence of HDAC2 (Fig. 2A). Most, if not all cellular HDAC1 and -2 is associated with higher-order pro- tein complexes in the nucleus (1). We used coimmunoprecipi- tation of individual components of the Sin3A, NuRD, and Fig. 2. Loss of HDAC1 results in decreased deacetylase activity associated CoREST complexes to assess the associated HDAC activity and with HDAC1/2 complexes and an increase H3K56Ac. (A) Quantitative West- their integrity in cells lacking either HDAC1 or HDAC2. In the ern blot of HDAC1, -2, and -3 obtained from nuclear extracts of the indicated cell types. Protein levels were quantitated using a LiCOR scanner and nor- absence of HDAC1 we observe a decrease in the deacetylase malized to the level of α-tubulin. Data represent independent experiments, activity associated with each of the Sin3A, NuRD, and CoREST using three independent clones. (*, P < 0.05; Student’s t test). Mean values complexes, with the largest reduction in the CoREST complex (n =3)± SEM are plotted. (B) Specific antisera to the indicated proteins were (Fig. 2B). This decrease in activity occurs despite the fact that we used to coimmunoprecipitate Sin3A, NuRD (αMTA-2), and CoREST (α-LSD1) detect increased amounts of HDAC2 in each of these same complexes from HDAC1Lox/Lox, HDAC1Δ2/Δ2, HDAC2Lox/Lox, and HDAC2Δ2/Δ2 ES complexes (Fig.