To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major ...cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
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•Persistent P300/CBP KAT activity is required to maintain oncogenic gene expression•P300/CBP inhibition modulates transcription independently of DNA accessibility•Compensatory loss of NCoR/SMRT complexes overcomes catalytic P300/CBP inhibition•Histone methylation switching imparts stable transcriptional suppression
Hogg et al. use multimodal genomics to explore the acute epigenetic and transcriptional effects of therapies targeting histone acetyltransferases P300/CBP in cancer. Catalytic P300/CBP inhibition selectively impairs expression of key oncogenic transcription factors, leading to robust anti-tumor responses, but remains inherently reversible prior to compensatory histone methylation switching.
Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and ...aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2 Delta mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2 Delta shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2 Delta strains. By strong SIRT1 overexpression in sir2 Delta cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2 Delta mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.
Expression of the estrogen receptor- alpha (ER alpha ) gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding ...the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ~400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of ESR1 gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2 gamma . Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of ESR1 mRNA expression that highlights the importance of the chromatin environment at the -150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells.
Background Mesenchymal stem cells (MSCs) have been considered as ideal cells for the treatment of a variety of diseases. However, aging and spontaneous differentiation of MSCs during culture ...expansion dampen their effectiveness. Previous studies suggest that ex vivo aging of MSCs is largely caused by epigenetic changes particularly a decline of histone H3 acetylation levels in promoter regions of pluripotent genes due to inappropriate growth environment. Methodology/Principal Findings In this study, we examined whether histone deacetylase inhibitor trichostatin A (TSA) could suppress the histone H3 deacetylation thus maintaining the primitive property of MSCs. We found that in regular adherent culture, human MSCs became flatter and larger upon successive passaging, while the expression of pluripotent genes such as Oct4, Sox2, Nanog, Rex-1, CD133 and TERT decreased markedly. Administration of low concentrations of TSA in culture significantly suppressed the morphological changes in MSCs otherwise occurred during culture expansion, increased their proliferation while retaining their cell contact growth inhibition property and multipotent differentiation ability. Moreover, TSA stabilized the expression of the above pluripotent genes and histone H3 acetylation levels in K9 and K14 in promoter regions of Oct4, Sox2 and TERT. Conclusions/Significance Our results suggest that TSA may serve as an effective culture additive to maintain the primitive feature of MSCs during culture expansion.
Energy band structure of inorganic nano-sonosensitizers is usually optimized by surface decoration with noble metals or metal oxide semiconductors, aiming to enhance interfacial charge transfer, ...augment spin-flip and promote radical generation. To avoid potential biohazards of metallic elements, herein, metal-free graphitic carbon nitride quantum dots (g-C
N
QDs) are anchored onto hollow mesoporous TiO
nanostructure to formulate TiO
@g-C
N
heterojunction. The direct Z-scheme charge transfer significantly improves the separation/recombination dynamics of electron/hole (e
/h
) pairs upon ultrasound (US) stimulation, which promotes the yield of singlet oxygen (
O
) and hydroxyl radicals (·OH). The conjugated g-C
N
QDs with peroxidase-mimic activity further react with the elevated endogenous H
O
and aggravate oxidative stress. After loading prodrug romidepsin (RMD) in TiO
@g-C
N
, stimulus-responsive drug delivery can be realized by US irradiation. The disulfide bridge of the released RMD tends to be reduced by glutathione (GSH) into a monocyclic dithiol, which arrests cell cycle in G2/M phase and evokes apoptosis through enhanced histone acetylation. Importantly, reactive oxygen species accumulation accompanied by GSH depletion is devoted to deleterious redox dyshomeostasis, leading to augmented systemic oncotherapy by eliciting antitumor immunity. Collectively, this paradigm provides useful insights in optimizing the performance of TiO
-based nano-sonosensitizers for tackling critical diseases.
STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to ...specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes: CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr's ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration (p = 0.0030, r2 = 0.0784). Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.
Chromatin is a highly compact and dynamic nuclear structure that consists of DNA and associated proteins. The main organizational unit is the nucleosome, which consists of a histone octamer with DNA ...wrapped around it. Histone proteins are implicated in the regulation of eukaryote genes and they carry numerous reversible post-translational modifications that control DNA-protein interactions and the recruitment of chromatin binding proteins. Heterochromatin, the transcriptionally inactive part of the genome, is densely packed and contains histone H3 that is methylated at Lys 9 (H3K9me). The propagation of H3K9me in nucleosomes along the DNA in chromatin is antagonizing by methylation of H3 Lysine 4 (H3K4me) and acetylations of several lysines, which is related to euchromatin and active genes. We show that the related histone modifications form antagonized domains on a coarse scale. These histone marks are assumed to be initiated within distinct nucleation sites in the DNA and to propagate bi-directionally. We propose a simple computer model that simulates the distribution of heterochromatin in human chromosomes. The simulations are in agreement with previously reported experimental observations from two different human cell lines. We reproduced different types of barriers between heterochromatin and euchromatin providing a unified model for their function. The effect of changes in the nucleation site distribution and of propagation rates were studied. The former occurs mainly with the aim of (de-)activation of single genes or gene groups and the latter has the power of controlling the transcriptional programs of entire chromosomes. Generally, the regulatory program of gene transcription is controlled by the distribution of nucleation sites along the DNA string.
Resveratrol, a natural polyphenol compound, has broad effects on critical events, including inflammation, oxidation, cancer and aging. However, the function and molecular mechanisms of resveratrol on ...T cell activation are controversial. In the present study, we found that resveratrol significantly inhibits the activation and cytokine production of T cells in vitro and in vivo. Sirt1 expression was up-regulated in resveratrol-treated T cells. Once Sirt1 was down-regulated in the T cells, the resveratrol-induced inhibition of T cell activation noticeably diminished. The acetylation of c-Jun decreased and its translocation was impeded in the resveratrol-treated T cells. The incidence and severity of collagen-induced arthritis in the resveratrol-treated mice were considerably reduced.
SUMMARY
Modification of the O‐acetylation level of xyloglucan (XyG) appears to affect aluminum (Al) sensitivity in Arabidopsis by modulating its binding capacity to Al. However, the transcriptional ...regulation of this process remains largely unknown. In our previous studies, we found that the expression of TRICHOME BIREFRINGENCE‐LIKE27 (TBL27), which is responsible for the O‐acetylation of XyG, was downregulated under Al stress. In the present study, we showed that the expression of an R2R3‐type transcription factor‐encoding gene, MYB103, was also inhibited by Al exposure and exhibited a co‐expression pattern with TBL27 in roots and siliques, suggesting a potential link between MYB103 and TBL27. The loss of function of MYB103 resulted in increased Al sensitivity, as indicated by more inhibited root growth and elevated root Al content compared with the wild type. Moreover, we also detected increased Al accumulation in the root cell wall and the hemicellulose fraction, which was attributed to the changes in the O‐acetylation level of XyG rather than the XyG content itself. In addition, further analysis revealed that MYB103 positively activated TBL27 expression by directly binding to the TBL27 promoter region, and TBL27 overexpression in the myb103 mutant rescued the Al‐sensitive phenotype of the mutant to the wild‐type level. Taken together, we conclude that MYB103 acts upstream of TBL27 to positively regulate Al resistance by modulating the O‐acetylation of the cell wall XyG.
Significance Statement
In this study, a member of the MYB family that is involved in the regulation of aluminum (Al) sensitivity in Arabidopsis, MYB103, was identified. Further analysis indicated that MYB103 acts upstream of TRICHOME BIREFRINGENCE‐LIKE27 in regulating Al sensitivity by modulating the O‐acetylation level of the cell wall xyloglucan.
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