Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells Shereen Jamaladdina,1, Richard D. W. Kellya,1, Laura O’Regana, Oliver M. Doveyb, Grace E. Hodsona, Christopher J. Millarda, Nicola Portolanoa, Andrew M. Frya, John W. R. Schwabea, and Shaun M. Cowleya,2 aDepartment of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom; and bWellcome Trust Sanger Institute, Hinxton, Cambs CB10 1SA, United Kingdom Edited by Mark Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved June 3, 2014 (received for review November 13, 2013) Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic modulation. In the process of DNA double-strand break repair, components of corepressor complexes that modulate gene ex- for instance, they deacetylate histones in the vicinity of the le- pression. In most cell types, deletion of both Hdac1 and Hdac2 is sion, which is a critical step in nonhomologous end joining (13). required to generate a discernible phenotype, suggesting their During DNA replication, new histones are deposited in an activity is largely redundant. We have therefore generated an ES acetylated form before being rapidly deacetylated as the chro- cell line in which Hdac1 and Hdac2 can be inactivated simulta- matin matures (14). A recent study that used the isolation of neously. Loss of HDAC1/2 resulted in a 60% reduction in total proteins on nascent DNA (iPOND) technique, found that HDAC activity and a loss of cell viability. Cell death is dependent HDAC1/2 are recruited close to the replication fork (15), which upon cell cycle progression, because differentiated, nonproliferat- suggests that they play a role in the maturation of chromatin ing cells retain their viability. Furthermore, we observe increased during S phase. Successful mitosis also requires appropriate mitotic defects, chromatin bridges, and micronuclei, suggesting patterns of histone acetylation. The Sin3A complex helps maintain HDAC1/2 are necessary for accurate chromosome segregation. pericentric heterochromatin in a hypoacetylated state to ensure correct assembly of the kinetochore and faithful chromosome Consistent with a critical role in the regulation of gene expression, CELL BIOLOGY microarray analysis of Hdac1/2-deleted cells reveals 1,708 differen- segregation (16, 17). HDAC1/2 are likely to be expressed in most, tially expressed genes. Significantly for the maintenance of stem if not all, mammalian cells. A generic requirement for HDAC1/2 cell self-renewal, we detected a reduction in the expression of the in cell cycle progression makes them potentially useful thera- pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. peutic targets in the treatment of cancer, because blocking their HDAC1/2 activity is regulated through binding of an inositol tet- activity with small-molecule inhibitors should limit cellular pro- liferation (11). raphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of With a few significant exceptions [embryogenesis (18), heart whether IP regulates the activity of the complex in cells. By res- (19), and brain (20)], the physiological activities of HDAC1 and 4 HDAC2 are functionally redundant. Conditional deletion of cuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate Significance that HDAC1/2 have essential and pleiotropic roles in cellular pro- liferation and regulate stem cell self-renewal by maintaining ex- Histone deacetylase 1 and 2 (HDAC1/2) are sister proteins that pression of key pluripotent transcription factors. regulate access to DNA by modulating chromatin. We have generated the first double knockout (DKO) of Hdac1/2 in em- istone deacetylase 1 and 2 (HDAC1/2) are highly related bryonic stem (ES) cells and find that gene inactivation causes Henzymes (∼80% identical) that regulate chromatin structure a loss of cell viability, which is associated with increased ab- as components of the multiprotein Sin3 (1, 2), NuRD (3), and normal mitotic spindles and chromosome segregation defects. CoREST (4) corepressor complexes. The recruitment and ac- Transcriptome analysis revealed that almost 2,000 genes are tivity of HDAC1/2 within these multiprotein complexes was deregulated in DKO cells. Significantly for the self-renewal thought to be a constitutive process. However, it has recently properties of ES cells, this includes down-regulation of the core pluripotent factors, Oct4, Nanog, and Rex1. Furthermore, using been shown that the HDAC1/metastasis-associated 1 (MTA1) Hdac1/2 complex (of NuRD) is regulated by a molecule of inositol tet- the rescue of -null cells as a model system to monitor HDAC1/2 activity, we have also shown that mutations that raphosphate [Ins(1,4,5,6)P ] (IP ), which regulates its enzymatic 4 4 abolish inositol tetraphosphate binding reduce the activity of activity, presumably in response to external signaling pathways HDAC1 in vivo. (5). Because deacetylation of histone tails results in the tight- ening of nucleosomal arrays (6), the physiological roles of Author contributions: S.J., R.D.W.K., L.O., O.M.D., A.M.F., J.W.R.S., and S.M.C. designed HDAC1/2 have mostly been defined within the context of tran- research; S.J., R.D.W.K., L.O., G.E.H., C.J.M., N.P., and S.M.C. performed research; O.M.D. scriptional repression. However, genome-wide mapping of HDAC1 and G.E.H. contributed new reagents/analytic tools; S.J., R.D.W.K., G.E.H., C.J.M., A.M.F., target genes reveals a correlation between binding and tran- J.W.R.S., and S.M.C. analyzed data; and S.J., R.D.W.K., O.M.D., and S.M.C. wrote the paper. scriptional activity (7, 8), suggesting that HDAC1/2 may play The authors declare no conflict of interest. a role in the cyclical acetylation of histones at active pro- This article is a PNAS Direct Submission. moters (9). HDAC1/2 may thus have roles in both transcrip- Freely available online through the PNAS open access option. tional activation and repression. Data deposition: The microarray data reported in this paper have been deposited in the Although there are a burgeoning number of proteins in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. acetylome in addition to histones (10), the Sin3A, NuRD, and GSE52134). CoREST complexes contain multiple DNA/chromatin recogni- 1S.J. and R.D.W.K. contributed equally to this work. tion motifs (11), which, in combination with transcription factors 2To whom correspondence should be addressed. E-mail: [email protected]. (2, 12), target HDAC1/2 to chromatin. Their physiological roles This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. should therefore be viewed within the framework of chromatin 1073/pnas.1321330111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1321330111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Hdac1 or Hdac2 alone, using tissue-specific transgenic models, produced no obvious deleterious effects on the development of heart, smooth muscle, endothelial cells, neural crest cells (19), oocytes (21), epidermis (22), B cells (23), and T cells (24); whereas simultaneous deletion of Hdac1/2 in these same cell types produced a number of profound phenotypes [summarized in Kelly and Cowley (11)]. We recently described the generation and characterization of conditional knockout embryonic stem (ES) cells for Hdac1 or Hdac2 (25). Although their differentia- tion properties are altered, cell viability and pluripotent potential of ES cells were unaffected by loss of either HDAC1 or HDAC2 alone. To circumvent this functional redundancy, we have engi- neered a double conditional knockout (DKO) (HDAC1Lox/Lox; HDAC2Lox/Lox; CreER) cell line, in which we can inactivate both genes simultaneously using a tamoxifen-inducible Cre/estrogen receptor fusion expressed from the ROSA26 locus. We demon- strate that loss of HDAC1/2 causes loss of cell viability 4 days following gene inactivation, which is associated with an increase in abnormal mitotic spindles and chromosome segregation defects. Almost 2,000 genes are deregulated. Significantly for the self-renewal properties of ES cells, this includes down-regulation of the core pluripotent factors, Oct4, Nanog, and Rex1. Fur- thermore, using the rescue of Hdac1/2-null cells as a model system to monitor HDAC1/2 activity, we have also shown for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1 in vivo. Results Inactivation of Hdac1/2 Causes Defective Chromosomal Segregation and a Loss of Cell Viability. We generated a conditional Hdac1Lox/Lox; Fig. 1. Inactivation of Hdac1/2 causes loss of cell viability. (A) Schematic Hdac2Lox/Lox;CreERDKO ES cell line, in which exon 2 of each diagram of the model system. An E14 ES cell line constitutively expressing A a Cre/estrogen receptor (CreER) fusion from the ROSA26 locus was used to gene is flanked by LoxP sites (Fig. 1 ), using standard gene tar- generate homozygous conditional knockout alleles for both Hdac1 and geting methodology. Induction of CreER, using 4-hydroxytamoxifen Hdac2. Both genes are inactivated by deletion of exon 2, which is flanked by (OHT), causes deletion of exon 2 and disrupts the ORF such that LoxP sites (blue triangles). (B) Quantitative Western blot showing loss of a premature stop codon is introduced in exon 3. Consistent with our HDAC1 and HDAC2 proteins following gene inactivation (0–4 d). Cells were previous data (25), inactivation of the Hdac1 and Hdac2 genes cultured in the presence of 4-hydroxytamoxifen (OHT) for 24 h to induce the resulted in loss of each protein 2–3 days after OHT treatment (Fig. deletion of Hdac1/2.