HDAC1 Depletion Research Articles

Cell Differentiation and Aging Lead To Up-Regulation of FTO, While the ALKBH5 Protein Level Was Stable During Aging but Up-Regulated During in vitro-Induced Cardiomyogenesis.

FTO and ALKBH5 proteins are essential erasers of N6-adenosine methylation in RNA. We studied how levels of FTO and ALKBH5 proteins changed during mouse embryonic development, aging, cardiomyogenesis, and neuroectodermal differentiation. We observed that aging in male and female mice was associated with FTO up-regulation in mouse hearts, brains, lungs, and kidneys, while the ALKBH5 level remained stable. FTO and ALKBH5 proteins were up-regulated during experimentally induced cardiomyogenesis, but the level of ALKBH5 protein was not changed when neuroectodermal differentiation was induced. HDAC1 depletion in mouse ES cells caused FTO down-regulation. In these cells, mRNA, carrying information from genes that regulate histone signature, RNA processing, and cell differentiation, was characterized by a reduced level of N6-adenosine methylation in specific gene loci, primarily regulating cell differentiation into neuroectoderm. Together, when we compared both RNA demethylating proteins, the FTO protein level undergoes the most significant changes during cell differentiation and aging. Thus, we conclude that during aging and neuronal differentiation, m6A RNA demethylation is likely regulated by the FTO protein but not via the function of ALKBH5.

The Highest Density of Phosphorylated Histone H1 Appeared in Prophase and Prometaphase in Parallel with Reduced H3K9me3, and HDAC1 Depletion Increased H1.2/H1.3 and H1.4 Serine 38 Phosphorylation.

Background: Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and apoptosis. Heterochromatinization, as the main feature of these processes, is also associated with pronounced trimethylation of histones H3 at the lysine 9 position (H3K9me3). Methods: By confocal microscopy, we analyzed cell cycle-dependent levels and distribution of phosphorylated histone H1 (H1ph) and H3K9me3. By mass spectrometry, we studied post-translational modifications of linker histones. Results: Phosphorylated histone H1, similarly to H3K9me3, has a comparable level in the G1, S, and G2 phases of the cell cycle. A high density of phosphorylated H1 was inside nucleoli of mouse embryonic stem cells (ESCs). H1ph was also abundant in prophase and prometaphase, while H1ph was absent in anaphase and telophase. H3K9me3 surrounded chromosomal DNA in telophase. This histone modification was barely detectable in the early phases of mitosis. Mass spectrometry revealed several ESC-specific phosphorylation sites of H1. HDAC1 depletion did not change H1 acetylation but potentiated phosphorylation of H1.2/H1.3 and H1.4 at serine 38 positions. Conclusions: Differences in the level and distribution of H1ph and H3K9me3 were revealed during mitotic phases. ESC-specific phosphorylation sites were identified in a linker histone.

Downregulation of TrkB Expression and Signaling by Valproic Acid and Other Histone Deacetylase Inhibitors.

Valproic acid (VPA) has been shown to regulate the levels of brain-derived neurotrophic factor (BDNF), but it is not known whether this drug can affect the neuronal responses to BDNF. In the present study, we show that in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells, prolonged exposure to VPA reduces the expression of the BDNF receptor TrkB at the protein and mRNA levels and inhibits the intracellular signaling, neurotrophic activity, and prosurvival function of BDNF. VPA downregulates TrkB and curtails BDNF-induced signaling also in differentiated Kelly and LAN-1 neuroblastoma cells and primary mouse cortical neurons. The VPA effect is mimicked by several histone deacetylase (HDAC) inhibitors, including the class I HDAC inhibitors entinostat and romidepsin. Conversely, the class II HDAC inhibitor MC1568, the HDAC6 inhibitor tubacin, the HDAC8 inhibitor PCI-34051, and the VPA derivative valpromide have no effect. In neuroblastoma cells and primary neurons both VPA and entinostat increase the cellular levels of the transcription factor RUNX3, which negatively regulates TrkB gene expression. Treatment with RUNX3 siRNA attenuates VPA-induced RUNX3 elevation and TrkB downregulation. VPA, entinostat, HDAC1 depletion by siRNA, and 3-deazaneplanocin A (DZNep), an inhibitor of the polycomb repressor complex 2 (PRC2), decrease the PRC2 core component EZH2, a RUNX3 suppressor. Like VPA, HDAC1 depletion and DZNep increase RUNX3 and decrease TrkB expression. These results indicate that VPA downregulates TrkB through epigenetic mechanisms involving the EZH2/RUNX3 axis and provide evidence that this effect implicates relevant consequences with regard to BDNF efficacy in stimulating intracellular signaling and functional responses. SIGNIFICANCE STATEMENT: The tropomyosin-related kinase receptor B (TrkB) mediates the stimulatory effects of brain-derived neurotrophic factor (BDNF) on neuronal growth, differentiation, and survival and is highly expressed in aggressive neuroblastoma and other tumors. Here we show that exposure to valproic acid (VPA) downregulates TrkB expression and functional activity in retinoic acid-differentiated human neuroblastoma cell lines and primary mouse cortical neurons. The effects of VPA are mimicked by other histone deacetylase (HDAC) inhibitors and HDAC1 knockdown and appear to be mediated by an epigenetic mechanism involving the upregulation of RUNX3, a suppressor of TrkB gene expression. TrkB downregulation may have relevance for the use of VPA as a potential therapeutic agent in neuroblastoma and other pathologies characterized by an excessive BDNF/TrkB signaling.

Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells.

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

HDAC1 Depletion in Human Cardiac Mesenchymal Stromal Cells Facilitates Paracrine‐Mediated Endothelial Cell Growth and Tube Formation through a Mechanism Involving Enhanced bFGF Production and Secretion

IntroductionCardiac mesenchymal stromal cell (CMC) administration has been documented to improve cardiac function in pre‐clinical animal models of heart failure. While the precise mechanism(s) underlying their therapeutic benefits remain unclear, both transdifferentiation (contributing to formation of new cardiac parenchyma) and secretion of paracrine signaling molecules (promoting neovascularization, cell survival, etc.) have been implicated as major modes by which transplanted cells exert their cardiac reparative effects. Thus, many laboratories have focused on novel methods to improve donor cell cardiogenic differentiation and/or cytokine secretion to enhance their therapeutic potential. We have previously shown either pharmacologic inhibition or genetic depletion of HDAC1 to promote CMC lineage commitment towards a cardiomyogenic/endothelial cell‐like fate. While cardiogenic differentiation of HDAC1‐inhibited CMCs may account for functional improvements, we have previously shown inhibition of HDAC activity to alter CMC cytokine secretion – an effect that may have profound consequences on endogenous repair mechanisms (including cell proliferation and neovascularization). To this end, in the current study, we sought to investigate the influence of HDAC1‐depletion on CMC cytokine secretion and associated paracrine‐mediated activities on endothelial cell function in vitro.MethodsPatient‐derived CMCs were transduced with shRNA constructs targeting human HDAC1 (shHDAC1) or non‐target (shNT) controls. Conditioned media (CM) was collected from shHDAC1 or shNT transduced CMCs cultured in F12 media in the absence of FBS for 24 h. Cytokine protein arrays were employed to comprehensively assess and compare/contrast the expression of >100 secreted proteins in CM from shHDAC1 or shNT‐transduced CMCs. In vitro functional assays for cell proliferation, protection from oxidative stress, cell migration, and tube formation were performed on human endothelial cells incubated with CM from untransduced, shNT, or shHDAC1 human CMCs to compare/contrast paracrine signaling activity.ResultsCytokine protein arrays revealed a pronounced increase in the secretion of a number of cytokines involved in cell growth, migration, and differentiation in CM from shHDAC1‐transduced CMCs. Consistent with these observations, shHDAC1 CM more efficiently promoted endothelial cell proliferation and tube formation compared to that of CM from shNT or untransduced CMCs. In an effort to narrow down which secreted factors may be responsible for these affects, key cytokines previously implicated in cell therapy‐mediated cardiac repair were interrogated in shHDAC1, shNT, and untransduced CMCs. We revealed bFGF to be significantly upregulated at both the mRNA and protein levels in shHDAC1‐transduced CMCs vis‐à‐vis shNT and untransduced CMCs. Furthermore, shRNA‐mediated depletion of bFGF in HDAC1‐depleted CMCs was able to inhibit the effects of shHDAC1 CM in promoting both endothelial proliferation and tube formation. Thus, our results demonstrate that HDAC1 depletion activates CMC proangiogenic paracrine signaling through a mechanism involving the enhanced secretion of bFGF.ConclusionThese results reveal a hitherto unknown role for HDAC1 in the modulation of CMC cytokine secretion and implicate the targeted inhibition of HDAC1 in CMCs as a means to enhance paracrine‐mediated neovascularization in cardiac cell therapy applications.

Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest

The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1–3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress.

Loss of histone deacetylase Hdac1 disrupts metabolic processes in intestinal epithelial cells

By using acetyl-CoA as a substrate, acetyltransferases and histone deacetylases regulate protein acetylation by adding or removing an acetyl group on lysines. Nuclear-located Hdac1 is a regulator of intestinal homeostasis. We have previously shown that Hdac1 define specific intestinal epithelial cell basal and inflammatory-dependent gene expression patterns and control cell proliferation. We show here that Hdac1 depletion in cellulo leads to increased histone acetylation after metabolic stresses, and to metabolic disturbances resulting in impaired responses to oxidative stresses, AMPK kinase activation and mitochondrial biogenesis. Thus, nuclear Hdac1 may control intestinal epithelial cell metabolism by regulating the supply of acetyl groups.

Depletion of histone deacetylase 1 inhibits metastatic abilities of gastric cancer cells by regulating the miR-34a/CD44 pathway.

Overexpression of histone deacetylases (HDACs) is associated with higher metastatic rates and a poor prognosis in gastric cancer. However, the underlying mechanisms involved remain unclear. The present study aimed to investigate the molecular pathways that are involved in HDAC1-mediated metastatic activities in gastric cancer cells. First we used a microRNA (miRNA or miR) microarray to screen potential miRNAs whose expression can be altered by HDAC1 depletion. Of these miRNAs, miR-34a is important as it is often inactivated in cancer cells and acts as a tumor suppressor for various types of cancer. The reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) results confirmed that miR-34a was upregulated by HDAC1 knockdown. Cells depleted of HDAC1 had lower abilities to migrate, invade and adhere, which were restored by a miR-34a antagomiR. Depletion of HDAC1 also resulted in impaired microfilaments and microtubules, while co-transfection of the miR-34a antagomiR attenuated these changes in the cellular cytoskeleton. The HDAC1/miR-34a axis regulated the expression and activation of CD44 and its downstream factors including Bcl-2, Ras homolog family member A (RhoA), LIM domain kinase 1 (LIMK-1) and matrix metalloproteinase (MMP)-2. The latter three proteins were responsible for the organization of tubulin and actin cytoskeleton and the formation of cellular pseudopodia. In conclusion, results of the present study indicated that HDAC1 depletion inhibits the metastatic abilities of gastric cancer cells by regulating the miRNA-34a/CD44 pathway, which may be a potential target for the treatment of gastric cancer.

The histone deacetylase Hdac1 regulates inflammatory signalling in intestinal epithelial cells

BackgroundIt has recently been found that both nuclear epithelial-expressed histone deacetylases Hdac1 and Hdac2 are important to insure intestinal homeostasis and control the mucosal inflammatory response in vivo. In addition, HDAC inhibitors modulate epithelial cell inflammatory responses in cancer cells. However, little is known of the specific role of different HDAC, notably Hdac1, in the regulation of inflammatory gene expression in intestinal epithelial cells (IEC).MethodsWe investigated the role of Hdac1 in non-transformed IEC-6 rat cells infected with lentiviral vectors expressing specific Hdac1 shRNAs, to suppress Hdac1 expression. Proliferation was assessed by cell counting. Deacetylase activity was measured with a colorimetric HDAC assay. Cells were treated with IL-1β and/or the JQ1 bromodomain acetyl-binding inhibitor. Nuclear protein levels of Hdac1, Hdac2, phosphorylated or unphosphorylated NF-κB p65 or C/EBPβ, and NF-κB p50 and actin were determined by Western blot. Chemokine and acute phase protein expression was assessed by semi-quantitative RT-PCR analysis. Secreted cytokine and chemokine levels were assessed with a protein array. Chromatin immunoprecipitation experiments were done to assess RNA polymerase II recruitment.ResultsReduced Hdac1 protein levels led to Hdac2 protein increases and decreased cell proliferation. Hdac1 depletion prolonged nuclear IL-1β-induced phosphorylation of NF-κB p65 protein on Ser536 as opposed to total p65, and of C/EBPβ on Ser105. In addition, semi-quantitative RT-PCR analysis revealed three patterns of expression caused by Hdac1 depletion, namely increased basal and IL-1β-stimulated levels (Hp, Kng1), increased IL-1β-stimulated levels (Cxcl2) and decreased basal levels with normal IL-1β induction levels (Ccl2, Ccl5, Cxcl1, C3). Secreted cytokine and chemokine measurements confirmed that Hdac1 played roles both as an IL-1β signalling repressor and activator. Hdac1 depletion did not alter the JQ1 dependent inhibition of basal and IL-1β-induced inflammatory gene expression. Hdac1 depletion led to decreased basal levels of RNA polymerase II enrichment on the Ccl2 promoter, as opposed to the Gapdh promoter, correlating with decreased Ccl2 basal mRNA expression.ConclusionsHdac1 is a major nuclear HDAC controlling IL-1β-dependent inflammatory response in IEC, notably by regulating gene-specific transcriptional responses. Hdac1 may be important in restricting basal and inflammatory-induced gene levels to defined ranges of expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-014-0043-2) contains supplementary material, which is available to authorized users.

Histone deacetylase 8 is required for centrosome cohesion and influenza A virus entry.

Influenza A virus (IAV) enters host cells by endocytosis followed by acid-activated penetration from late endosomes (LEs). Using siRNA silencing, we found that histone deacetylase 8 (HDAC8), a cytoplasmic enzyme, efficiently promoted productive entry of IAV into tissue culture cells, whereas HDAC1 suppressed it. HDAC8 enhanced endocytosis, acidification, and penetration of the incoming virus. In contrast, HDAC1 inhibited acidification and penetration. The effects were connected with dramatic alterations in the organization of the microtubule system, and, as a consequence, a change in the behavior of LEs and lysosomes (LYs). Depletion of HDAC8 caused loss of centrosome-associated microtubules and loss of directed centripetal movement of LEs, dispersing LE/LYs to the cell periphery. For HDAC1, the picture was the opposite. To explain these changes, centrosome cohesion emerged as the critical factor. Depletion of HDAC8 caused centrosome splitting, which could also be induced by depleting a centriole-linker protein, rootletin. In both cases, IAV infection was inhibited. HDAC1 depletion reduced the splitting of centrosomes, and enhanced infection. The longer the distance between centrosomes, the lower the level of infection. HDAC8 depletion was also found to inhibit infection of Uukuniemi virus (a bunyavirus) suggesting common requirements among late penetrating enveloped viruses. The results established class I HDACs as powerful regulators of microtubule organization, centrosome function, endosome maturation, and infection by IAV and other late penetrating viruses.

Differential effects of class I isoform histone deacetylase depletion and enzymatic inhibition by belinostat or valproic acid in HeLa cells

BackgroundHistone acetylation is an epigenetic modification involved in the regulation of gene expression, balanced by histone acetyl transferases and histone deacetylase (HDAC) enzymes. HDAC inhibitors (HDACi) induce growth arrest and cell death in transformed cells, and are currently in many clinical cancer trials. The transcriptional response to HDACi is complex, as is the response to HDAC isoform knockdown (KD). Here, we describe for the first time in a human cancer cell line, a transcriptional comparison of treatment by two structurally unrelated HDACi; belinostat and valproic acid with the KD of HDAC1, 2 and 3 isoforms.ResultsHDAC KD showed anti-proliferative effects, although to a lesser extent than HDACi treatment. Moreover, we found a 2-fold increased resistance of HDAC1 knockdown cells to belinostat, suggesting this isoenzyme as a selective target. While both HDACi treatment and individual class I HDAC KD produce significant transcriptional effects, three-times higher for HDACi, the gene-expression profiles of class I HDAC KD compared with that obtained by HDACi treatment exhibited less than 4% of altered genes in common between the two modes of inhibition. Further, cell-specific effects of HDAC KD are evident by comparison with a recent study in a different cell line.ConclusionThe increased resistance to belinostat in response to HDAC1 depletion indicates the possibility of using this isoform as a predictive biomarker of response to HDACi treatment. Further, the transcriptional response to chemical inhibition of HDACs is very different from that of KD of individual class I HDAC isoforms. These data suggest that the anti-tumor effect of HDACi is indeed linked to class I inhibition, but may be more complex than simply targeting individual HDAC enzymes.

Parthenolide Specifically Depletes Histone Deacetylase 1 Protein and Induces Cell Death through Ataxia Telangiectasia Mutated

Histone deacetylases (HDACs), enzymes involved in chromatin remodeling, are promising targets for anticancer drug development. Several HDAC inhibitors (HDACi) are in clinical trials. One limitation of present HDACi is their nonspecificity, affecting many HDACs with similar effectiveness. We have identified a small molecule, the sesquiterpene lactone parthenolide (PN), which specifically depletes HDAC1 protein without affecting other class I/II HDACs. HDAC1 depletion occurred through proteasomal degradation and resulted in transcriptional consequences comparable to those observed with pan-HDACi. Surprisingly, HDAC1 depletion did not occur through the inflammation mediator IKK2, a known PN target and regulator of HDAC1. Rather, PN promoted HDAC1 depletion and cell death through the DNA-damage-transducer ataxia telangiectasia mutated. Our study suggests that modulating cellular HDAC protein levels with small molecules provides an alternative approach to specific HDAC inhibition and effective cancer treatment.

Tumour necrosis factor‐α depletes histone deacetylase 1 protein through IKK2

Class I histone deacetylases (HDACs) are ubiquitous enzymes that repress gene expression by deacetylating histone tails and promoting chromatin compaction. Pro-inflammatory agents activate programmes of gene expression through transcription factors such as nuclear factor-kappaB (NF-kappaB), even in the context of ubiquitous HDAC activity. How this is accomplished remains unknown. We found that cells treated with the pro-inflammatory cytokine tumour necrosis factor-alpha rapidly and substantially reduced HDAC1 protein levels without affecting other class I HDACs. In addition, HDAC1 depletion occurred through protein degradation, required IKK2 activity and resulted in increased transcription from both NF-kappaB-associated and unassociated gene promoters. Our study suggests that the activation of programmes of gene expression by pro-inflammatory agents requires global changes in specific critical epigenetic regulators such as HDAC1.