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Intracellular calcium is related to cardiac hypertrophy. The CaV1.2 channel and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaM regulate the intracellular calcium content. However, the differences in CaMKII and CaM in cardiac hypertrophy are still conflicting and are worthy of studying as drug targets. Therefore, in this study, we aim to investigate the roles and mechanism of CaM and CaMKII on CaV1.2 in pathological myocardial hypertrophy. The results showed that ISO stimulation caused SD rat heart and cardiomyocyte hypertrophy. In vivo, the HW/BW, LVW/BW, cross-sectional area, fibrosis ratio and ANP expression were all increased. There were no differences in CaV1.2 channel expression in the in vivo model or the in vitro model, but the ISO stimulation induced channel activity, and the [Ca2+]i increased. The protein expression levels of CaMKII and p-CaMKII were all increased in the ISO group, but the CaM expression level decreased. AIP inhibited ANP, CaMKII and p-CaMKII expression, and ISO-induced [Ca2+]i increased. AIP also reduced HDAC4, p-HDAC and MEF2C expression. However, CMZ did not play a cardiac hypertrophy reversal role in vitro. In conclusion, we considered that compared with CaM, CaMKII may be a much more important drug target in cardiac hypertrophy reversal.

Hyperglycemia promotes podocyte apoptosis and plays an important role in the pathogenesis of diabetic nephropathy (DN). Calcium/calcineurin (CaN) signaling is critical for podocyte apoptosis. Therefore, it is essential to elucidate the mechanisms underlying the regulation of CaN signaling. Recent studies reported that histone deacetylase 4 (HDAC4) is involved in podocyte apoptosis in DN. The aim of this study was to determine whether HDAC4 mediates the regulation of CaN and to elucidate the function of HDAC4 in high glucose (HG)-induced podocyte apoptosis. First, we identified the expression of HDAC4 was upregulated in podocytes of patients with DN. In vitro, the results also indicate that the mRNA and protein expression levels of HDAC4 were increased in HG-cultured podocytes. Silencing and overexpression of HDAC4 markedly decreased and increased CaN expression, respectively. Meanwhile, HG-induced podocyte apoptosis was abrogated by HDAC4-knockdown with subsequent decreased Bax expression and increased Bcl-2 expression. In contrast, overexpression of HDAC4 increased podocyte apoptosis and Bax expression, as well as decreased Bcl-2 expression. In addition, podocyte apoptosis induced by HDAC4 overexpression was effectively rescued by FK506, a pharmacological inhibitor of CaN, which was accompanied by decreased Bax and increased Bcl-2 expression. As a novel finding, HG-induced podocyte apoptosis is mediated by the HDAC4/CaN signaling pathway, which presents a promising target for therapeutic intervention in DN.

Background Rheumatoid arthritis (RA) is a common immune-related disease worldwide, which is characterized by impaired fibroblast-like synoviocytes (FLS) proliferation and increased release of inflammatory cytokines. Unfortunately, the detailed mechanism by which miR-138-modulated rheumatoid arthritis has not been fully understood. Methods RT-qPCR was used to examined mRNA level of various genes and western blot was utilized to probe protein level of acetylated H3, p-p62 and IκBα. For cytokines detection, we used ELISA method to measure the extracellular level of these cytokines. Bioinformatic tool and dual-luciferase reporter assay were employed to predict and confirm the downstream target of miR-138. Results miR-138 was upregulated in serum and synovial tissues of RA patients. Moreover, Increased miR-138 was observed in LPS-treated FLS cells. HDAC4 was shown as the direct target of miR-138 and could be negatively regulated by miR-138. miR-138 and HDAC4 were involved in RA-related inflammatory cytokines release of FLS cells. Next, we revealed NF-κB and PGRN were significantly modulated by HDAC4 and miR-138 in an acetylation-dependent manner. More importantly, IκBα depletion and PGRN overexpression had the ability to rescue miR-138 inhibitor-attenuated inflammatory cytokines release of FLS cells. Conclusion Here, we reveal miR-138 regulates RA-related inflammatory cytokines in rheumatoid arthritis through HDAC4/PGRN or HDAC4/NF-κB. Our findings uncover a new molecular mechanism implicated in rheumatoid arthritis, which may accelerate development of therapeutical strategy by targeting this mechanism.

Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.

Blood pressure is regulated not only by peripheral arterial resistance, but also by heart, kidney, and central nervous system. We have previously demonstrated that expression level of calmodulin-related proteins including eukaryotic elongation factor 2 kinase (eEF2K), death-associated protein kinase (DAPK)3, and histone deacetylase (HDAC)4 was specifically elevated in mesenteric artery from spontaneously hypertensive rats (SHR), which partly contributes to the development of hypertension via vascular inflammation and structural remodeling. We tested the hypothesis whether expression and localization of eEF2K, DAPK3, and HDAC4 are altered in brain, heart, and kidney from SHR. After brain, left ventricles (LV), and kidney were isolated from 12-week-old male Wistar Kyoto rats (WKY) and SHR, Western blotting and histological analysis were performed. In brain tissue, protein expression of eEF2K and HDAC4 was abundant, whereas DAPK3 protein was less. HDAC4 protein expression in SHR brain was significantly higher than that in WKY brain. In LV, protein expression of eEF2K was relatively higher than DAPK3 or HDAC4, and it was significantly higher in SHR than WKY. In kidney tissue, protein expression of DAPK3 was the highest and seemed to be localized specifically to renal tubule. The present results indicate that the increased HDAC4 in brain and increased eEF2K in LV might be at least in part related to the development of hypertension.

Our previous study showed that the level of glutamate carboxypeptidase II (GCPII) protein is regulated by valproic acid, a histone deacetylase (HDAC) inhibitor, through acetylation of lysine residue in the GCPII protein in human astrocytes, U-87MG. The present study further investigated which HDAC subtype is involved in the acetylation of GCPII. The results revealed that GCPII interacted with HDAC1 but not with HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6. Overexpression of catalytic domain (1-56 aa)-deleted HDAC1, which poorly binds to GCPII, enhanced lysine acetylation in GCPII and increased the level of GCPII protein when compared with that of the wild-type HDAC1. Further experiments showed that HDAC1 regulated the stability of GCPII protein. These data suggest that acetylation of GCPII is facilitated by HDAC1, and the acetylated GCPII is more stable than the non-acetylated GCPII. Additional experiments using siRNA HDAC1 and by HDAC1 overexpression confirmed the role of HDAC1 in regulating the stability of GCPII protein. Further, database search of acetylation and ubiquitination sites showed four candidate lysine sites in human GCPII protein that can be both acetylated and ubiquitinylated (K207, K479, K491, and K699). Mutation (lysine residues to arginine (R)) analysis showed that in the presence of cycloheximide K479R- and K491R-hGCPII mutants were less ubiquitinylated and degraded, and decrease in the level of GCPII protein by HDAC1 was significantly blocked by K479R mutants. These data suggest that K479 is a possible site of acetylation or ubiquitination. Furthermore, the results also demonstrate that the stability of GCPII protein is regulated by HDAC1 through acetylation at the lysine 479 residue.

BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

Cell fate determination is tightly controlled by the expression of transcription factors and gene regulatory networks. PAX6 is a transcription factor containing a DNA-binding paired-box domain and homeobox domain that plays a key role in the development of the eye, brain, and pancreas. Here, we showed that histone deacetyltransferase 1 (HDAC1) is a novel binding partner of PAX6 in newborn mouse retinas. We also showed that HDAC1 specifically binds to the paired and transactivation domains of PAX6, and these physical interactions were required for effective repression of PAX6 transcriptional activity during retinal development. Furthermore, HDAC1 preferentially regulates the transcriptional activity of PAX6 when it binds to paired-domain (P6CON and chimeric pCON/P3) PAX6 responsive elements compared to homeodomain (pP3) PAX6 responsive elements. The repressive effect of HDAC1 on the transcriptional activity of PAX6 was reversed by knockdown of HDAC1 or treatment with an HDAC inhibitor, TSA. Taken together, these results show that HDAC1 binds PAX6 and that protein-protein interaction leads to transcriptional repression of PAX6 target genes during mouse retinal development.

Sirt7 localizes in the nucleus (enriched in the nucleolus) and is an NAD(+)-dependent deacetylase with high selectivity for the acetylated lysine 18 of histone H3 (H3K18Ac). It has been reported that Sirt7 is necessary for maintaining the fundamental properties of the cancer cell phenotype and stabilizing the tumorigenicity of human cancer via deacetylation of H3K18Ac. However, the regulators of Sirt7 deacetylase activity are unknown. Myb-binding protein 1a (Mybbp1a) is reported to interact with and regulate the function of a number of transcription factors. In the present study, we demonstrated that Mybbp1a binds to Sirt7 in vitro and in vivo. Serial deletion studies indicated that N- and C-terminal regions of Sirt7 and C-terminal region of Mybbp1a are important for the binding. Furthermore, transfection experiments showed that Mybbp1a is capable of inhibiting the deacetylation activity of H3K18Ac by Sirt7. Our findings demonstrate that Mybbp1a is a novel negative regulator of Sirt7.

Calmodulin (CaM) is associated with a variety of cell functions including inflammation, apoptosis, and muscular contraction. It is recently clarified that some CaM-related proteins are responsible for cardiovascular diseases. We therefore explored CaM-related proteins that mediate hypertensive vascular diseases. Expression levels of six CaM-related proteins with almost unknown function in blood vessels were examined in aorta and mesenteric artery from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) by Western blotting. In aorta from SHR, eukaryotic elongation factor (eEF)2 kinase (eEF2K) and death-associated protein kinase (DAPK)3 protein increased compared with WKY, while Ca(2+)/CaM-dependent protein kinase IIδ, histone deacetylases (HDAC)4 and HDAC5 protein decreased. In mesenteric artery from SHR, eEF2K, HDAC4 and DAPK3 protein increased compared with WKY, while HDAC5 decreased. Our findings demonstrate that expression levels of several CaM-related proteins are changed in vascular tissues of SHR and suggest that CaM-related proteins might be at least in part related to the pathogenesis of hypertensive vascular diseases.

Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of alpha-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-beta1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against alpha-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-beta1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

The relationship between histone acetylation and transcriptional activation at the yeast INO1 gene was addressed. INO1 encodes a key enzyme required for the de novo synthesis of phosphatidylinositol. Induction of INO1 resulted in acetylation of both histones H3 and H4 at the INO1 promoter and sequences farther downstream in the coding region, suggesting a gene-wide acetylation in response to transcriptional activation. Such chromatin remodeling activity requires the presence of transcriptional activator Ino2p. This indicates that histone acetylation is an activator-dependent event. Furthermore, the increase of histone acetylation is due to the increase of acetylation levels per nucleosome rather than the increase of nucleosome density. Therefore, these observations constitute evidence for the molecular mechanism of the correlation between histone acetylation and INO1 activity.

It has been well documented that histone deacetylase inhibitors suppress inflammatory gene expression. Therefore, we investigated whether histone deacetylase inhibitors modulate the expression of caspase-11 that is known as an inducible caspase regulating both inflammation and apoptosis. In the present study, we show that sodium butyrate and trichostatin A, two structurally unrelated inhibitors of histone deacetylase (HDAC), effectively suppressed the induction of caspase-11 in mouse embryonic fibroblasts stimulated with lipopolysaccharides. Sodium butyrate inhibited the activation of upstream signaling events for the caspase-11 induction such as activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, degradation of inhibitor of {kappa}B, and activation of nuclear factor-{kappa}B. These results suggest that the HDAC inhibitor suppressed cytosolic signaling events for the induction of caspase-11 by inhibiting the deacetylation of non-histone proteins.

Histone deacetylases (HDACs) are subdivided into three classes—HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.

LEF-1 and E2F are both transcription factors involved in cell proliferation, differentiation and apoptosis. The present study shows for the first time that LEF-1 associates with E2F1 and further beta-catenin independently activates the E2F-responsive reporter gene by attenuating the interaction between E2F1 and Histone deacetylase 1 (HDAC1), which indicates that LEF-1, except for its function in Wnt signaling, may play a distinct role via activating the transcription of E2F1.

The Aryl hydrocarbon receptor repressor (AhRR) has been proposed to inhibit Aryl hydrocarbon receptor (AhR) activity by competing with AhR for forming a heterodimer with AhR nuclear translocator (Arnt) and subsequently binding to the xenobiotic responsive elements (XRE). However, the precise mechanism of AhRR inhibitory activity remains unknown. Analysis of the inhibitory activity of AhRR on the expression of a TK promoter-driven reporter has localized a core repressor domain in the sequence of amino acid residue 555-701. The inhibitory activity of AhRR is sensitive to a histone deacetylase (HDAC) inhibitor, trichostatin A. By using the yeast two-hybrid screening method with the C-terminal sequence of AhRR as bait, we identified a binding partner, Ankyrin-repeat protein2 (ANKRA2), a protein known to interact with HDAC4 and HDAC5. RNA interference experiments using ANKRA2 and AhRR siRNAs indicate that ANKRA2 is important for transcriptional repression by AhRR. We have found that under normal conditions, CYP1A1 gene is kept silent in MEF cells by AhRR/Arnt heterodimer, which binds to the XRE sequence in its promoter and recruits ANKRA2, HDAC4, and HDAC5 as co-repressors.

SUZ12 is a Polycomb group protein that forms Polycomb repressive complexes (PRC2/3) together with EED and histone methyltransferase EZH2. Although the essential role of SUZ12 in regulating the activity of the PRC2/3 complexes has been demonstrated, additional function of this protein was suggested. Here, we show that SUZ12 interacts with WD-repeat protein MEP50 in vitro and in vivo. We show that the MEP50 binds histone H2A selectively among core histones, and mediates transcriptional repression of protein arginine methyltransferase PRMT5, which is known to methylate H2A and H4. These results suggest that SUZ12 might have a role in transcriptional regulation through physical interaction with MEP50 that can be an adaptor between PRMT5 and its substrate H2A.

Histone deacetylase 3 (HDAC3) has been implicated to play roles in governing cell proliferation. Here we demonstrated that the overexpression of HDAC3 repressed transcription of p15{sup INK4b} and p21{sup WAF1/cip1} genes in 293T cells, and that the recruitment of HDAC3 to the promoter regions of these genes was critical to this repression. We also showed that HDAC3 repressed GAL4-Sp1 transcriptional activity, and that Sp1 was co-immunoprecipitated with FLAG-tagged HDAC3. We conclude that HDAC3 can repress p15{sup INK4b} and p21{sup WAF1/cip1} transcription by interacting with Sp1. Furthermore, knockdown of HDAC3 by RNAi up-regulated the transcriptional expression of p15{sup INK4b}, but not that of p21{sup WAF1/cip1}, implicating the different roles of HDAC3 in repression of p15{sup INK4b} and p21{sup WAF1/cip1} transcription. Data from this study indicate that the inhibition of p15{sup INK4b} and p21{sup WAF1/cip1} may be one of the mechanisms by which HDAC3 participates in cell cycle regulation and oncogenesis.

Histone acetylation is associated with transcriptional activation of many genes. However, the role of acetylation in transcriptional regulation of heat shock protein genes (hsp) still remains an obscure issue. Here we examined the effects of histone deacetylase inhibitors (HDIs), trichostatin A, and sodium butyrate, on changes in acetylation level of core histones and on expression of hsp22 gene in Drosophila melanogaster. The results showed that both HDIs elevated the acetylation level of histone H3. By using the chromatin immunoprecipitation, we located the HDI-induced H3 hyperacetylation at both the promoter and the downstream of RNA polymerase II of the transcribing hsp22 gene. Meanwhile, the elevated acetylation level increased the accessibility of heat shock factor to target cis-acting regulatory sites. We conclude that histone acetylation stimulates the transcription initiation and promotes the transcription elongation, thereby up-regulating both basal and inducible expression of hsp22 in D. melanogaster.

The acetylation status of histones plays an essential role in regulating transcription and replication, and is thus involved in the proliferation and differentiation of normal and neoplastic cells. Here, we investigated the effect of trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs), on G2–M transition during the cell cycle. HDAC inhibition by TSA arrested the cell cycle at G2 and also induced escape from the mitotic arrest into G1. TSA reduced the expression of cyclin B1, a key cyclin for G2–M transition, but stimulated expression of p21WAF1/Cip1, an inhibitor of CDK and Cdc2. In contrast, the expression of cyclin B1 but not p21WAF1/Cip1 is enhanced during M. Moreover, histone acetylation at promoters of these two genes was regulated by TSA. TSA augmented acetylation of the p21WAF1/Cip1 promoter but reduced that of the cyclin B1 promoter, suggesting the relationship between TSA-induced modulation of histone acetylation and differential expression of these genes. Taken together, our observations suggest that modulation of HDAC activity is implicated in the G2–M transition by regulating the transcription of cell cycle regulators, p21WAF1/Cip1 and cyclin B1, via modulating acetylation status of the histones at their promoters.

Epigenetically mediated modulation of gene promoter function through histone acetylation modifying enzymes, which regulate the acetylation state of histone proteins and other promoter-bound transcription factors, is increasingly appreciated as a key component in the regulation of reversible gene expression. While histone acetyltransferases (HATs), which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, histone deacetylases (HDACs) tend to associate with multisubunit corepressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. We have isolated and characterized the human HDAC9 genomic sequence, which spans a region of 458 kb and which has one single chromosomal locus. Determination of the exon–intron splice-junctions established that HDAC9 is encoded by 23 exons ranging in size from 22 bp (exon 1) to 264 bp (exon 11). Characterization of the 5 ′ flanking genomic region revealed that the human HDAC9 promoter lacks both the canonical TATA and CCAAT boxes; CpG elements are missing. The human HDAC9 open reading frame is 3036 bp long and encodes a 1011 aa protein with a predictive molecular weight of 111.3 kDa and an isoelectric point of 6.41. Fluorescence in situ hybridization analysis localized the human HDAC9 gene to chromosome 7p21, a region which has been associated particularly with the pathogenesis of gynecological tumors.

Histone acetylation has been shown to affect chromatin structure and gene expression. The mitogen-activated protein (MAP) kinase pathway is activated by a number of cytokines and plays critical roles in hematopoietic cell survival, proliferation, and differentiation. We focused on the part of the MAP kinase cascade and granulocyte colony-stimulating factor (G-CSF)in histone acetylation at one of the critical myeloid differentiation-associated genes, myeloperoxidase (MPO). G-CSF caused rapid acetylation of histone H3 and H4 at the promoter of MPO as revealed by chromatin immunoprecipitation. In addition, CBP and p300 were recruited to the promoter in response to G-CSF. Furthermore, we showed that rapid histone acetylation induced by G-CSF is MAP kinase-dependent. These results illustrate how myeloid-differentiating signals via G-CSF may be coupled with histone acetylation during the process of gene expression.

Apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-l-2-amino-8-oxodecanoyl)], a novel histone deacetylase inhibitor, has been identified as an antiprotozoal and antiproliferative agent. In this study, we show apicidin induces transcriptional activation of p21(WAF1/CIP1) (p21) in human prostate carcinoma cells. Apicidin induces expression of p21 protein and mRNA and activation of p21 promoter-luciferase reporter constructs. Apicidin causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin. Chromatin immunoprecipitation shows p21 promoter DNA is associated with hyperacetylated histones H3 and H4 after treatment with apicidin. Therefore, the data here demonstrate that apicidin activates p21 transcription associated with the acetylation of histones H3 and H4.

MBP-1, a c-myc promoter binding protein, is a mammalian transcription factor with intriguing properties including transcriptional repression of cellular genes. Recently, we have identified and characterized two different repressor domains of MBP-1. In this report, we have demonstrated that MBP-1 physically associates with histone deacetylase (HDAC), thus promoting formation of neucleosomes that inhibit transcription. Trichostatin A, an inhibitor of histone deacetylase, significantly reduces MBP-1-mediated transcriptional repression. However, MBP-1-mediated repression on c-myc promoter is resistant to histone deacetylase activity. Our results suggest that MBP-1 represses transcription by recruiting histone deacetylase as one of the mechanisms, whereas the other mechanism is resistant to HDAC activity and probably related to direct binding of promoter sequences or interaction through yet unidentified factor.

Reversible histone acetylation plays a central role in X chromosome inactivation. Histones are hypoacetylated on the heavily methylated inactive X chromosome and are hyperacetylated in the unmethylated "CpG islands" in animal genomes. We have investigated whether histone acetylation is involved in the regulation of the allelic expression of insulin-like growth factor II (IGF2), a maternally imprinted gene. HSK09, a human fibroblast cell line, retained normal monoallelic expression of IGF2 in culture. When these cells were treated with histone deacetylase inhibitors, sodium butyrate or trichostatin A, biallelic expression of IGF2 was observed from all of the promoters that are expressed. These results suggest that, in addition to DNA methylation, differential histone acetylation of two parental alleles may be another potential mechanism by which the imprinting of IGF2 is regulated, probably through changes in the local chromatin structure of the imprinted locus on chromosome 11p15.

Flowering time is tightly controlled by several regulatory pathways including photoperiod, vernalization in which epigenetic processes are involved. In this work, we have found that the Arabidopsis histone deacetylase gene HDA9 is involved in flowering time control. Mutation of the gene led to an early flowering phenotype in short day grown plants while without effect in long days. Analysis of flowering time regulatory gene expression revealed that hda9 mutations highly induced the expression of AGL19, but had no effect on CO, SOC1 or FLC. Chromatin immunoprecipitation assays indicated that the mutations led to a clear increase of histone H3K9 and H3K27 acetylation on the AGL19 gene in short days. AGL19 promotes flowering in a way independent of the CO and FLC pathways and has been shown to be repressed by polycomb group repressive complex2 (PRC2) EMF2 but activated by vernalization. The induced levels of AGL19 expression and histone acetylation by the hda9 mutations were comparable to that of the gene under long day conditions, indicating that AGL19 is regulated also by day length and that HDA9 is involved in short day repression of AGL19 by promoting histone H3 deacetylation, which may be related to the PRC2 EMF2 complex.

Lhx2, a member of LIM homeobox transcription factors, plays a key role in central nervous system (CNS) and embryonic tissue development. However, molecular mechanism of Lhx2 gene regulation remains largely unknown. Here, we identified and characterized a regulatory region of Lhx2 gene which mediates responses to two different signals such as inhibition of HDAC3 and stimulation by E2F1. In particular, the promoter region of −229 to −126 was responsible not only for basal expression but also for a inhibitor of histone deacetylase, trichostatin A (TSA)-mediated activation of Lhx2 gene. Intriguingly, transcription factor E2F1 also activates Lhx2 gene via direct binding to the same −229 to −126 region. Based on these observations, we could have demonstrated that E2F1 is necessary for TSA-mediated activation of Lhx2 gene and acetylation of histone 3 is involved in this event. This study provides evidence that the histone modification and E2F1 binding are integral parts of the mechanism for Lhx2 gene expression.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are three δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene and an underlying mechanism indicated selective interference with the HDAC4-MEF2 signaling pathway. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target.

Histone deacetylase inhibitors (HDACIs) are promising anti-tumor agents that selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Fundamentally, HDACIs are proposed to function by activating the transcription of genes, including the potent cyclin dependent kinase inhibitor p21 WAF1 . However, HDACIs primarily increase p21 WAF1 expression at the post-transcriptional level in HepG2 cells, implying that these anti-tumor agents regulate genes at multiple levels. Here, two novel cis -acting elements in the 3′ untranslated region (UTR) of p21 WAF1 are identified that control the ability of HDACIs to induce p21 WAF1 mRNA stabilization. Collectively, these studies highlight the complexity of HDACIs in gene regulation.

The sirtuins (SIRT1–7), also being referred to as class III HDACs, exert NAD-dependent deacetylase and/or ADP-ribosyltransferase activities in various cellular compartments including the cell nucleus, the cytoplasm and the mitochondria. The sirtuins play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. SIRT4 is a mitochondrial protein that lacks deacetylase activities but efficiently works as an ADP-ribosyltransferase. We have isolated and characterized the murine Sirt4 genomic sequence, which spans a region of 12 kb and which has one single genomic locus. Determination of the exon–intron splice junctions established that SIRT4 is encoded by 6 exons. The 1648 bp murine Sirt4 transcript encodes a 418 aa protein with a predictive molecular weight of 47.3 kDa. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt4 gene on chromosome 5F and is neighbored by the PLA2G1B and PXN genes.

Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.

Histone deacetylases (HDACs) form HDAC-associated complexes and play an essential role in transcriptional repression. The functional significance of HDAC-associated proteins in the progression of the cell cycle and in cell death remains to be established. Here, we investigated the molecular mechanisms by which methyl CpG-binding domain protein 3 (MBD3), a component of the HDAC complex, modulates these processes via its functional interplay with HDAC. Depletion of MBD3 induced an arrest at the G(2)/M transition and resulted in defective mitosis in cancer cells. These effects appear to be associated with the transcriptional modulation of key cell cycle-regulator genes, including CylinB1, Plk1, and Survivin. Chromatin immunoprecipitation analyses revealed that the transcription of these cell cycle regulators is modulated by MBD3, supporting its direct role in their transcriptional repression. These findings collectively support a role for MBD3 in cell cycle progression and cell death as a modulator of HDAC-mediated transcription.

Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a manner dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.

Changes on histone H3 modifications from methylation at lysine 9 to acetylation at lysine 9/14 is thought to be important for transcriptional activation of genes associated with differentiation. In this study, we found that the treatment with a p44/42 MAPK inhibitor, PD98059, induced di-methylation at lysine 9 on the upstream/transcriptional regions of the GLUT5 gene. Co-treatment of PD98059 with a glucocorticoid hormone agonist, dexamethasone (Dex), not only repressed the induction of di-methylation by PD98059, but also enhanced the acetylation of histone H3 at lysine 9/14 on the GLUT5 gene, as well as its gene expression. These results suggest that the histone H3 di-methylation at lysine 9, as well as acetylation at lysine 9/14, may be indispensable for coordinated induction of the GLUT5 gene by p44/42 MAP kinase inhibition and the glucocorticoid hormone.

Vascular endothelial growth factor receptors (VEGFRs) perform pivotal roles in both tumor growth and angiogenesis. In this study, we report that histone deacetylase inhibitors (HDIs) induce a reduction in VEGFR1 and VEGFR2 protein expression via the inhibition of class II histone deacetylases (HDACs) in human cancer cell lines. After HDI treatment, VEGFR1 and VEGFR2 were shown to be downregulated in a proteasome-dependent manner. HDI treatment induced a reduction in the binding of heat shock protein (Hsp) 90 to VEGFR1 or VEGFR2, followed by an increase of the binding of Hsp70 to VEGFR1 or VEGFR2. However, we noted no remarkable changes in the binding of Hsp90/Hsp70 to VEGFR3. HDI treatment effectively inhibited the activities of HDAC6 and HDAC10. Furthermore, the knock-down of HDAC6 or HDAC10, which was accomplished via the siRNA transfection, induced depletion of VEGFR1 or VEGFR2 proteins. Overall, these results indicate that HDAC6 and HDAC10 play important roles in Hsp-mediated VEGFR regulation.

Histone Deacetylase 1 (HDAC1) is a transcriptional regulator associated with proliferation, apoptosis, and tumorigenesis, although its precise cellular role is unclear. HDAC1 was previously characterized as a phosphoprotein where mutation of phosphorylated S421 and S423 resulted in a loss of deacetylase activity and protein association. Here, the role of phosphorylation in regulating HDAC1 function was examined using phospho-specific antibodies. The antibody studies revealed that phosphorylation at S421 and S423 is constant during the cell cycle, under stress conditions, or in the presence of kinase or phosphatase inhibitors. Further, phosphorylation is dispensable for catalysis or protein association in vitro, as revealed by phosphatase studies. Truncation mutants of HDAC1 demonstrated that binding to Sin3A is promoted by S421 and S423 phosphorylation, while interaction with RbAp48 is not. Taken together, the data are consistent with constitutive phosphorylation of HDAC1 at S421 and S423 in vivo, which is dispensable for activity in vitro.

Histone deacetylase (HDAC) inhibitors are one of promising drugs to induce fetal hemoglobin (HbF) for treatment of sickle cell disease (SCD) and beta-thalassemia. The HDAC inhibitor apicidin was recently reported as a powerful inducer of HbF via a mechanism involving p38 signaling. In this study, we further investigated the signaling effects on the transcriptional activation of gamma-globin gene. First, we compared histone 3 (H3) acetylation patterns of approximately 70kb beta-globin loci in K562 erythroid versus HeLa cells upon apicidin treatment by chromatin immunoprecipitation assays. The results showed that the level of H3 acetylation was globally increased from the LCR to the promoter of gamma-globin gene in K562 cells, but not in non-erythroid, HeLa cells. Inhibition of p38 signaling blocks the effects of apicidin-induced gamma-globin expression and H3 acetylation. In parallel, we assessed the recruitment of transcriptional complex to beta-globin locus following apicidin treatment. The binding of GATA-1, Sp1 and RNA polymerase II (pol II) were observed to increase over several regulatory regions of beta-globin locus. Inhibitor study revealed that p38 pathway was not involved in their recruitments by apicidin. Collectively, our results provide a molecular basis to elucidate the underlying mechanisms involving p38 signaling pathway in the inducement of gamma-globin transcriptional expression by apicidin.

The IL-2 gene expression is highly T-cell specific in an activation-dependent manner. However, little is known about how T-cell specific expression is regulated, although related transcription factors have been identified. To address this issue, we examined the chromatin structure change of the IL-2 gene by analyzing histone acetylation status in the upstream of IL-2 gene transcription initiation site. Interestingly, the histone acetylation level was found to be higher in various sites on the widespread upstream region in resting T-cells than resting B-cells. After T-cell stimulation with PMA and ionomycin, the same regions were further acetylated on histone H3. Particularly, the distal enhancer region showed prompt enhancement of histone acetylation, followed by the IL-2 mRNA expression. These results suggest that the 5' flanking region including the distal enhancer region of the IL-2 gene is already accessible in T cells with constitutive acetylation of histone H3,which may serve for T-cell specific IL-2 expression.

Histone deacetylase (HDAC) inhibitors have shown promise in clinical cancer therapy and to consistently induce p21WAF1/CIP1 expression in a p53-independent manner and via increased acetylation of the chromatin at the Sp1 sites in the p21WAF1/CIP1 promoter region. However, the exact mechanism by which HDAC inhibitors induce p21WAF1/CIP1 remains unclear. In this study, we observed that Trichostatin A (TSA), a HDAC inhibitor, induced strikingly p21WAF1/CIP1 expression in human cervical cancer (HeLa) cells, and this induction correlated with downregulation of c-myc expression. Coincident with this observation, knock down of c-myc with a c-myc specific small interfering RNA dramatically induced expression of p21WAF1/CIP1 in these cancer cells. These data suggest that c-myc may play a critical role in repression of p21WAF1/CIP1 expression in HeLa cells. More importantly, using chromatin immunoprecipitation assay, we observed for the first time that c-myc bound to the endogenous p21WAF1/CIP1 promoter in untreated HeLa cells, but not in TSA-treated cells. Taken together, TSA induced c-myc downregulation and release from the endogenous p21WAF1/CIP1 promoter contributes, at least partially, to transcriptional activation of the p21WAF1/CIP1 in HeLa cells.

Repression of gene transcription is mediated by histone deacetylases containing repressor-co-repressor complexes, which are recruited to promoters of target genes via interactions with sequence-specific transcription factors. The mammalian Sin3A co-repressor complex contains a core of at least seven proteins including the pRb-interacting protein RBP1 and a putative tumor suppressor p33(ING1b). By biochemical purification and mass spectrometry, we have identified a novel component p40 from this complex. p40 bears homology to both yeast Sds3, a component of yeast histone deacetylase complexes, and its mammalian homologue mSds3. The p40-associated complex purified from human cells shows a strong histone deacetylase activity. When tethered to a Gal-DNA binding domain, the Gal-p40 is able to significantly repress transcription of a Gal-luciferase promoter. Interestingly, database analysis reveals that p40 is also highly homologous to BRMS1, a breast carcinoma metastasis suppressor, and overexpression of p40 in human cells can significantly inhibit cell growth. Thus, our data indicate that p40 may be critically involved in transcription repression of cell growth-associated gene expression by recruiting the HDAC1 deacetylase complex.

Yin Yang 1 (YY1) is a highly conserved and multifunctional transcription factor. The diverse activities of YY1 are regulated and sometimes modified by interaction with various other proteins. By using a yeast two-hybrid screening system, SAP30 was identified as a protein that associates with YY1 and it is able to enhance YY1-mediated repression in a dose-dependent manner. SAP30 is a 30 kDa nuclear protein and is a component of the human histone deacetylase complex. In this study, the interaction of SAP30 and YY1 was confirmed both by in vitro and in vivo assays. The interaction domains between YY1 and SAP30 were mapped to the C-terminal segment of YY1 (295–414) and the C-terminal 91 amino acid region of SAP30. The observation that YY1, SAP30, and HDAC1 form a complex in vivo provides evidence that YY1 also recruits HDAC1 indirectly via its binding to SAP30. These results describe a novel mechanism for YY1-mediated repression.

In a chicken B cell line, DT40, the disruption of HDAC2 (chHDAC2) gene causes an alteration of several gene expressions including chicken IgM light chain (chIgM-L) gene by 2D-PAGE analysis. To investigate the transcriptional function of chHDAC2, we employed the chIgM-L promoter reporter plasmid. We found that chHDAC2 represses activated chIgM-L promoter activity. In transient expression experiments in NIH 3T3 cell, the specific histone deacetylase inhibitor tricostatin A (TSA) increased transactivation of chIgM-L promoter activity mediated by chicken Oct-1 and OBF-1 proteins. In transient coexpression of the three class I chicken histone deacetylases (chHDAC1-3) tested, only chHDAC2 repressed the activated chIgM-L promoter activity. These findings suggest that chHDAC2 might be recruited to the chIgM-L promoter and specifically repress chIgM-L transcription.

Histone acetylation is suggested to regulate gene expression in the TCR loci. Using R2CD3 mouse model, we previously showed that germline transcription of the TCRbeta chain gene is discrete between 5' Vbeta regions and Dbeta-Jbeta-Cbeta clusters plus Vbeta14 region. In this study, we investigated a role of histone H3 acetylation in transcriptional regulation of the TCRbeta locus. Our results showed that DN-DP transition is accompanied by significant promotion of histone H3 acetylation in both Dbeta-Jbeta-Cbeta cluster and Vbeta14 region, correlating with up-regulation of germline transcription. Surprisingly, termination of germline transcription of the 5' Vbeta regions was inversely correlated with histone H3 hyperacetylation. Moreover, histone H3 acetylation showed equivalent level in both functionally rearranged Vbeta region with active transcription and unrearranged Vbeta region without transcription in mature T cells. These results suggest that histone acetylation is not a sole limiting factor in both terminating germline Vbeta transcription during DN-DP transition and maintaining functionally rearranged Vbeta gene expression.

To better understand the link between chromatin modification and manganese superoxide dismutase (Mn-SOD) gene expression, we have investigated the level of histone acetylation at Mn-SOD proximal promoter. TSA induced the expression of Mn-SOD mRNA and its transcriptional activity in C2C12 cells. Sp1 binding sites in the proximal promoter region of Mn-SOD were transcriptionally responsive to TSA by transfection studies. We have detected a localized acetylation of histones H3 and H4, in vivo occupation by Sp1, early growth responsive-1 (Egr-1), and histone deacetylase-1 (HDAC1) in the proximal promoter region of Mn-SOD gene using chromatin immunoprecipitation assays. Our findings indicate that Mn-SOD gene expression is repressed by Sp1–HDAC1 complex. This repression is released by a localized histone acetylation and at least in parts a displacement by Egr-1 in response to TSA.

Histone deacetylase (HDAC) inhibitors have been demonstrated to regulate myeloid cell differentiation. In the present study the effects of the HDAC inhibitor trichostatin A (TSA) on the tetraspanin cell surface antigen CD9 were determined in primary murine macrophages. TSA inhibited CD9 protein and message expression and was optimal by 48 h. TSA did not induce similar effects on other surface markers and resulted in a modest increase or no effect on CD54 and CD11b, respectively. These effects were concentration dependent and concomitant with increased histone H4 acetylation. While interferon-gamma (IFN-gamma) and TSA had similar effects on CD9 expression, transcriptional profiling demonstrated significant differences in the genes activated by these stimuli. Notably CD14 message was down-regulated by IFN-gamma while increased by TSA. These results demonstrate that HDAC inhibition may modulate macrophage function in part through changes in the expression of membrane proteins associated with matrix interactions.

As a result of screen searching for proteins interacting with MEF-2 transcription factor, we have identified the hda-7 gene in Caenorhabditis elegans. The hda-7 locus encodes a class II histone deacetylase containing a highly conserved catalytic domain. C. elegans HDA-7 protein translated in vitro demonstrated a direct interaction with CeMEF-2, as shown in other organisms. CeHDA-7 is abundantly expressed in body-wall muscle cells, neurons, and hypodermal seam cells, similar to CeMEF-2 expression patterns. Consistent with previously known phenotypes observed in mef-2 deletion mutants [Dev. Biol. 223 (2000) 431], RNA interference targeted for hda-7 did not result in muscle function or developmental defects.

Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation. The acetylation of core histone proteins is reversible and regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increasing evidence suggests a connection between histone acetylation and the development of cancer and leukemia. We have recently mapped HDAC3 to mouse chromosome 18B3, a region which is syntenic with human chromosome 5q31, where HDAC3 is imbedded in a group of potential tumor suppressor genes and which has been reported to be the smallest commonly deleted segment in malignant myeloid disease. We report herein the identification and characterization of HDAC3, a yeast RPD3 ortholog in the mouse. Studies on murine HDAC3 may yield important insights on the understanding of myeloproliferative disease in humans.