Histone deacetylases (HDACs) modulate chromatin structure and transcription, but little is known about their function in mammalian development. HDAC1 was implicated previously in the repression of genes required for cell proliferation and differentiation. Here we show that targeted disruption of both HDAC1 alleles results in embryonic lethality before E10.5 due to severe proliferation defects and retardation in development. HDAC1-deficient embryonic stem cells show reduced proliferation rates, which correlate with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1). Similarly, expression of p21 and p27 is up-regulated in HDAC1-null embryos. In addition, loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 and concomitant changes in other histone modifications. The expression of HDAC2 and HDAC3 is induced in HDAC1-deficient cells, but cannot compensate for loss of the enzyme, suggesting a unique function for HDAC1. Our study provides the first evidence that a histone deacetylase is essential for unrestricted cell proliferation by repressing the expression of selective cell cycle inhibitors.

The chromatin of eukaryotic cells is organized in nucleosomes. This organization allows the efficient packaging of chromosomal DNA into the nucleus but limits the access of highmolecular-weight protein complexes of the transcription machinery. At least two different mechanisms enable the eukaryotic cell to relieve nucleosomal repression: the chromatinremodeling complexes (reviewed in references 55 and 57) and reversible histone acetylation. Two recent reports indicate a direct link between these two activities (60, 67). Posttranslational acetylation on conserved lysine residues within the Nterminal regions of nucleosomal histones is assumed to lead to a reduced attraction between chromosomal DNA and histone tails and changed interactions with neighboring nucleosomes or other nonhistone proteins. The resulting local chromatin decondensation increases the accessibility of particular DNA regions for RNA polymerase complexes. Consistent with this idea, transcriptionally active chromatin correlates with histone hyperacetylation (reviewed in references 18, 30, 47, 49, 61, and 62). This model predicts that histone acetyltransferases would promote transcription, while histone deacetylases (HDACs) should act as repressors. In accordance with this model, several transcriptional adapters and coactivators, such as GCN5 (8, 31), p300/CBP (4, 46), TAFII250 (40), SRC-1 (54), and ACTR (10), have been classified as histone acetyltransferases. Five HDACs have been identified in mammalian cells (12,14,56,58,63,64). Three of them, HDAC1, HDAC2, and HDAC3, have significant homology to yeast Rpd3 (44,50,59). HDAC4 and HDAC5 belong to the histone deacetylase A (HDA) family (9, 58). HDAC1 and HDAC2 are found in high-molecularweight complexes associated with adapter proteins like SIN3, SAP18, and SAP30 and nuclear corepressors like N-CoR, SMRT, and 24,32,42,65,66). Recently it was demonstrated that several mammalian transcription factors, such as Mad (21, 24, 32, 52), YY1 (64), hormone-dependent nuclear receptors (24, 42), MeCP2 (26, 43), CBF (27), retinoblastoma protein (Rb) (7, 38, 39), and related pocket proteins (16), can repress transcription by recruiting HDACs to specific promoters. In addition, the aberrant recruitment of HDACs by PLZF, PML, and ETO fusion proteins can interfere with the differentiation of hematopoietic precursor cells in acute promyelocytic leukemia (13,17,19,35).In this study we investigated the potential function of HDACs as transcriptional repressors during the growth arrest of mammalian cells. Using the S-phase-specific mouse thymidine kinase (TK) promoter as a model system, we show that HDAC1 can mediate transcriptional repression via the Sp1 binding site. HDAC1 is associated with Sp1 and binds directly to the C-terminal part of Sp1 that was previously identified as interacting domain for E2F1 (28). Sp1 and E2F1 cooperate in the activation of S-phase-specific promoters (28, 36). Here we show that E2F1 but not E2F4 can compete with HDAC1 binding to Sp1, thereby relieving HDAC1-mediated repression of the TK...

The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.The tumor suppressor p53 can induce cell cycle arrest or apoptosis in response to a variety of stress signals, such as DNA damage, oncogenic stimuli, or hypoxia (reviewed in reference 49). Activation of p53 occurs by several mechanisms including protein stabilization and modification of the protein by phosphorylation and acetylation. p53 is a transcription factor that recognizes specific binding sites within numerous target genes including mdm2, cyclin G, bax, and p21/WAF1/CIP1 (for reviews see references 5 and 12). While multiple downstream targets are involved in the mediation of apoptotic effects, the main target for p53-induced cell cycle arrest seems to be the p21 gene. p21 has been identified by virtue of its activation by p53 (13), its association with cyclin/cyclin-dependent kinase (CDK) complexes (23, 66), and its up-regulation during senescence (47). Furthermore, the p21 protein was shown previously to interact with the proliferating cell nuclear antigen (PCNA), thereby preventing DNA replication (10). Induction of p21 expression by genotoxic stress and its role during terminal differentiation of various cell types have been investigated intensively. While p21 is activated by p53-dependent mechanisms in response to DNA damage to ensure cell cycle arrest and repair, a variety of agents that promote differentiation, like phorbol ester or okadaic acid, can up-regulate p21 independently of p53 (for a review see reference 16). Similarly, the p21 gene can be activated by transforming growth factor ␤, Ca 2ϩ , lovastatin, or nerve growth factor (16).Recently, a number of reports demonstrated the induction of p21 by inhibitors of histone deacetylases (HDACs), such as sodium butyrate (46), trichostatin A (TSA) (56), suberoylanilide hydroxamic a...

Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression, and developmental events. The acetylation state of histones is controlled by the activities of acetylating and deacetylating enzymes. By using differential mRNA display, we have identified a mouse histone deacetylase gene, HD1, as an interleukin-2-inducible gene in murine T cells. Sequence alignments revealed that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator. Indirect immunofluorescence microscopy proved that mouse HD1 is a nuclear protein. When expressed in yeast, murine HD1 was also detected in the nucleus, although it failed to complement the rpd3⌬ deletion phenotype. HD1 mRNA expression was low in G 0 mouse cells but increased when the cells crossed the G 1 /S boundary after growth stimulation. Immunoprecipitation experiments and functional in vitro assays showed that HD1 protein is associated with histone deacetylase activity. Both HD1 protein levels and total histone deacetylase activity increased upon interleukin-2 stimulation of resting B6.1 cells. When coexpressed with a luciferase reporter construct, HD1 acted as a negative regulator of the Rous sarcoma virus enhancer/promoter. HD1 overexpression in stably transfected Swiss 3T3 cells caused a severe delay during the G 2 /M phases of the cell cycle. Our results indicate that balanced histone acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells.

The amino-terminal tails of core histones are targets for multiple modifications such as acetylation, phosphorylation, and methylation. Generation of modification-specific antibodies and the identification of some of the modifying enzymes allow us to begin to understand the impact of these modifications on several cellular processes, including DNA replication and transcription. Reversible histone acetylation emerged during recent years as an important mechanism for the chromatindependent regulation of gene expression. Acetylation of ε-amino groups of lysine residues results in reduced interaction between positively charged histone tails and negatively charged DNA. Local or wide-range histone deacetylation leads to chromatin condensation, while acetylation is believed to increase the accessibility of particular genomic regions for high-molecular-weight protein complexes, thereby setting the stage for transcription.In addition to acetylation, histone phosphorylation has been recently shown to play an important role for chromatin-associated processes. Distinct sets of kinases have been implicated in these events (references 4 and 41 and references cited therein). On one hand, H3 phosphorylation correlates with entry into mitosis, suggesting a link between chromatin condensation and histone modification by kinases. On the other hand, histone H3 phosphorylation at serine 10 was found to be an important step of the so-called nucleosomal response (26; reviewed in reference 41). This term describes the phosphorylation of histone H3, which leads to the concomitant activation of the immediate-early genes c-fos, c-jun, and c-myc (3, 26). The nucleosomal response can be induced through stimulation of the mitogen-activated protein (MAP) kinase cascade by growth factors, pharmacological agents, or stress. Induction of the MAP kinase pathway leads to the activation of effector kinases (MSK1/Rsk-2) which can phosphorylate histone H3 (33,40).Only a small fraction of histone H3 is transiently phosphorylated at the G 0 /G 1 transition in growth factor-stimulated cells (1). This subset of phosphorylated histone H3 proteins is highly susceptible to hyperacetylation induced by histone deacetylase (HDAC) inhibitors. One possible explanation for this finding is the strong preference in in vitro experiments of several acetylating enzymes for histone H3 phosphorylated at serine 10 (5, 24). Indeed, a number of recent observations strongly suggest the presence of cross talk between the different histone modifying mechanisms (reviewed in references 19, 35, and 43). A link between histone acetylation and phosphorylation is provided by studies reporting the association of simultaneously acetylated and phosphorylated (in this report referred to as "phosphoacetylated") histone H3 with nucleosomes of activated immediate-early genes (5, 6, 23). A concerted action of acetyltransferases and kinases was also demonstrated in yeast (25) and in mammalian cells (28).An alternative model predicts the independent targeting of histone H3 by kinases and ac...

The avian erythroblastosis virus (AEV) oncoprotein v-ErbA represents a mutated, oncogenic thyroid hormone receptor α (c-ErbA/ TRα). v-ErbA cooperates with the stem cell factor-activated, endogenous receptor tyrosine kinase c-Kit to induce self-renewal and to arrest differentiation of primary avian erythroblasts, the AEV transformation target cells. In this cooperation, v-ErbA substitutes for endogenous steroid hormone receptor function required for sustained proliferation of non-transformed erythroid progenitors. In this paper, we propose a novel concept of how v-ErbA transforms erythroblasts. Using culture media strictly depleted from thyroid hormone (T3) and retinoids, the ligands for c-ErbA/TRα and its coreceptor RXR, we show that overexpressed, unliganded c-ErbA/ TRα closely resembles v-ErbA in its activity on primary erythroblasts. In cooperation with ligand-activated c-Kit, c-ErbA/ TRα causes steroidindependent, long-term proliferation and tightly blocks differentiation. Activation of c-ErbA/ TRα by physiological T3 levels causes the loss of self-renewal capacity and induces synchronous, terminal differentiation under otherwise identical conditions. This T3-induced switch in erythroid progenitor development is correlated with a decrease of c-ErbA-associated histone deacetylase activity. Our results suggest that the crucial role of the mutations activating v-erbA as an oncogene is to 'freeze' c-ErbA/ TRα in its non-liganded, repressive conformation and to facilitate its overexpression.

Proliferation and cell cycle progression of eukaryotic cells are closely linked to changes in chromatin structure and gene expression. By reversible histone acetylation the cell is able to modulate chromatin condensation and accessibility of specific regions within the chromatin. Here, we examined histone H4 acetylation patterns during growth induction of the murine interleukin-2 dependent T cell line B6.1. In order to detect acetylation on each of the four potential target residues we produced a set of antibodies recognizing specifically acetylated lysine 5, 8, 12 and 16 in the N-terminal tail of histone H4. Acetylation was generally low in resting T cells, but increased after stimulation with a specific kinetics for each lysine. Lysine 16 was acetylated during the G1 phase and deacetylated during S phase. H4 acetylation on lysine 5, 8 and 12, in contrast, was induced before cells started to replicate, and persisted until cells entered mitosis. Treatment of resting B6.1 cells with the specific deacetylase inhibitor trichostatin A (TSA) led to H4 hyperacetylation at all four lysine residues indicating that the histone modification can occur in the absence of replication. After release from TSA treatment normal H4 acetylation levels were reestablished by extremely rapid deacetylation of lysines 5, 8, 12 and 16. The deacetylation step was 60^100 times faster than TSA induced acetylation and equally efficient in resting and exponentially growing T cells. Our results indicate the presence of cell cycle regulated lysine specific acetylating and deacetylating activities in mouse T cells.z 1998 Federation of European Biochemical Societies.