Histone Acetyltransferase P300 Research Articles

Bromodomain protein 4 is a key molecular driver of TGFβ1-induced hepatic stellate cell activation.

Liver fibrosis is characterized by the excessive deposition of extracellular matrix in liver. Chronic liver injury induces the activation of hepatic stellate cell (HSCs), a key step in liver fibrogenesis. The activated HSC is the primary source of ECM and contributes significantly to liver fibrosis. TGFβ1 is the most potent pro-fibrotic cytokine. Bromodomain protein 4 (BrD4), an epigenetic reader of histone acetylation marks, was crucial for profibrotic gene expression in HSCs. The present study aimed to investigate the roles of BRD4 in TGFβ1-dependent HSC activation and liver fibrosis, focusing on TGFβ1-induced alterations of the levels of the fibrotic-related important proteins in HSCs by employing the heterozygous TGFβ1 knockout mice and BrD4 knockdown in vivo and in vitro. Results revealed that BrD4 protein level was significantly upregulated by TGFβ1 and BrD4 knockdown reduced TGFβ1-induced HSC activation and liver fibrosis. BrD4 was required for the influences of TGFβ1 on PDGFβ receptor and on the pathways of Smad3, Stat3, and Akt. BrD4 also mediated TGFβ1-induced increases in histone acetyltransferase p300, the pivotal pro-inflammatory NFkB p65, and tissue inhibitor of metalloproteinase 1 whereas BrD4 reduced Caspase-3 protein levels in HSCs during liver injury, independent of TGFβ1. Further experiments indicated the interaction between TGFβ1-induced BrD4 and NFkB p65 in HSCs and in liver of TAA-induced liver injury. Human cirrhotic livers were demonstrated a parallel increase in the protein levels of BrD4 and NFkB p65 in HSCs. This study revealed that BrD4 was a key molecular driver of TGFβ1-induced HSC activation and liver fibrosis.

Predicted Role of Acetyl-CoA Synthetase and HAT p300 in Extracellular Lactate Mediated Lactylation in the Tumor: In vitro and In silico Models

Background: As per the Warburg effect, cancer cells are known to convert pyruvate into lactate. The accumulation of lactate is associated with metabolic and epigenetic reprogramming, which has newly been suggested to involve lactylation. However, the role of secreted lactate in modulating the tumor microenvironment through lactylation remains unclear. Specifically, there are gaps in our understanding of the enzyme responsible for converting lactate to lactyl-CoA and the nature of the enzyme that performs lactylation by utilizing lactyl-CoA as a substrate. It is worth noting that there are limited papers focused on metabolite profiling that detect lactate and lactyl-CoA levels intracellularly and extracellularly in the context of cancer cells. Methods: Here, we have employed an in-house developed vertical tube gel electrophoresis (VTGE) and LC-HRMS assisted profiling of extracellular metabolites of breast cancer cells treated by anticancer compositions of cow urine DMSO fraction (CUDF) that was reported previously. Furthermore, we used molecular docking and molecular dynamics (MD) simulations to determine the potential enzyme that can convert lactate to lactyl-CoA. Next, the histone acetyltransferase p300 (HAT p300) enzyme (PDB ID: 6GYR) was evaluated as a potential enzyme that can bind to lactylCoA during the lactylation process. Results: We collected evidence on the secretion of lactate in the extracellular conditioned medium of breast cancer cells treated by anticancer compositions. MD simulations data projected that acetyl-CoA synthetase could be a potential enzyme that may convert lactate into lactyl-CoA similar to a known substrate acetate. Furthermore, a specific and efficient binding (docking energy -9.6 kcal/mol) of lactyl-CoA with p300 HAT suggested that lactyl-CoA may serve as a substrate for lactylation similar to acetylation that uses acetyl-CoA as a substrate. Conclusion: In conclusion, our data provide a hint on the missing link for the lactylation process due to lactate in terms of a potential enzyme that can convert lactate into lactyl-CoA. This study helped us to project the HAT p300 enzyme that may use lactyl-CoA as a substrate in the lactylation process of the tumor microenvironment.

Therapeutic Targeting of JAK/STAT Signaling and Histone Acetyltransferase Activity in Post-Myeloproliferative Neoplasm Secondary AML

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling is present in 50-90% of MPNs, underscoring its role in driving MPN pathogenesis and transformation to acute sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. Despite enhancing patients' quality of life, ruxolitinib did not exert major improvements on disease outcome (Eghtedar et al, 2012). Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CREBBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML using high throughput genetic screens, in vitro functional assays, transcriptomic and epigenetic profiling and mouse xenograft in vivo experiments. To identify druggable determinants of JAK2 inhibitor response in post-MPN sAML, we performed genome-wide loss-of-function CRISPR screens in sAML HEL cells treated with four different JAK2 inhibitors, including ruxolitinib. We discovered that depletion of the histone acetyl transferase CREBBP sensitizes HEL cells to JAK2 inhibition (Figure 1A). Pharmacological inhibition of JAK/STAT signaling and HAT activity using a combination of ruxolitinib and CREBBP inhibitors significantly reduced the proliferation of sAML cells HEL and SET2 (Figure 1B). Among the CREBBP inhibitors, CBP30 synergizes best with ruxolitinib having Chou-Talalay combination indices of 0.152 and 0.704 in HEL and SET2, respectively. Moreover, combination treatment of ruxolitinib and CBP30 markedly increased apoptosis and induced cell cycle arrest at G1 in HEL. To mechanistically characterize the synergism between ruxolitinib and CBP30, we performed ChIP-seq to profile STAT5 binding and H3K27ac levels in HEL cells treated with DMSO, ruxolitinib, CBP30 and their combination. STAT5 binding profiles did not reveal major differences in ruxolitinib versus ruxolitinib + CBP30 treated cells, suggesting that the drug combination does not significantly alter STAT5 binding. Conversely, we noticed striking global changes in H3K27ac levels in the combination compared with DMSO or single agents, indicating that the synergistic effect between ruxolitinib and CBP30 is mediated by substantial changes in transcriptional regulation. Notably, analysis of H3K27ac downregulated and upregulated sites identified enrichments in motifs recognized by FOS/SMAD4 and GATA transcription factors, respectively. These TFs are involved in the regulation of myeloid differentiation implicating a synergistic regulation of relevant myeloid differentiation pathways by the combination of JAK2 and CREBBP inhibitors in sAML. Furthermore, ruxolitinib + CBP30 treatment in HEL elicits a unique transcriptome profile with expression deregulation of genes associated with cell cycle, DNA repair, HAT activity and TGF-beta signaling as determined by DAVID functional annotation analysis. Lastly, to evaluate the synergistic effects of ruxolitinib and CBP30 in vivo, we treated luciferase-expressing HEL sAML mouse xenografts with vehicle, ruxolitinib (80 mg/kg), CBP30 (30 mg/kg) and ruxolitinib (80 mg/kg) + CBP30 (30 mg/kg). Combination of ruxolitinib and CBP30 displayed significant decrease in leukemia burden after 4 weeks of treatment as monitored by bioluminescence imaging and quantification. Consistently, mice treated with both drugs also showed remarkable decrease in their white blood cell count and spleen size compared with single-agent-treated and vehicle-treated mice. Collectively, our results substantiate the combinatorial therapeutic targeting of JAK/STAT signaling and HAT activity as a potential treatment approach for post-MPN sAML.

LINC00355 promotes gastric carcinogenesis by scaffolding p300 to activate CDC42 transcription and enhancing HNRNPA2B1 to stabilize CDC42 mRNA dependent on m6A.

LINC00355 is involved in the tumorigenesis of several types of cancer. We verified that LINC00355 is upregulated in gastric cancer (GC) and contributes to GC cells' proliferation and metastasis. RNA sequencing (RNA-seq) and rescue assays suggested that LINC00355 controls gastric carcinogenesis by regulating the expression of cell division cycle 42 (CDC42) guanosine triphosphatase (GTPases), thereby activating their downstream pathways. Most previous studies have shown that LINC00355 acts as a ceRNA by sponging miRNAs to modulate downstream gene expression. Our group focus on epigenetic regulatory potential of LINC00355 in gene expression. Mechanistically, LINC00355 binds to p300 histone acetyltransferase, specifying the histone modification pattern on the CDC42 promoter to activate CDC42 transcription, thereby altering GC cell biology. In addition, HNRNPA2B1, which is upregulated by LINC00355, recognizes the N6-methyladenosine (m6A) sites of CDC42 and enhances the stability of CDC42 mRNA transcripts. Therefore, LINC00355 is mechanistically, functionally, and clinically oncogenic in GC cells.

Suppression of DNMT2/3 by proinflammatory cytokines inhibits CtBP1/2-dependent genes to promote the occurrence of atrophic nonunion

Failure of bone healing after fracture often results in nonunion, but the underlying mechanism of nonunion pathogenesis is poorly understood. Herein, we provide evidence to clarify that the inflammatory microenvironment of atrophic nonunion (AN) mice suppresses the expression levels of DNA methyltransferases 2 (DNMT2) and 3A (DNMT3a), preventing the methylation of CpG islands on the promoters of C-terminal binding protein 1/2 (CtBP1/2) and resulting in their overexpression. Increased CtBP1/2 acts as transcriptional corepressors that, along with histone acetyltransferase p300 and Runt-related transcription factor 2 (Runx2), suppress the expression levels of six genes involved in bone healing: BGLAP (bone gamma-carboxyglutamate protein), ALPL (alkaline phosphatase), SPP1 (secreted phosphoprotein 1), COL1A1 (collagen 1a1), IBSP (integrin binding sialoprotein), and MMP13 (matrix metallopeptidase 13). We also observe a similar phenomenon in osteoblast cells treated with proinflammatory cytokines or treated with a DNMT inhibitor (5-azacytidine). Forced expression of DNMT2/3a or blockage of CtBP1/2 with their inhibitors can reverse the expression levels of BGLAP/ALPL/SPP1/COL1A1/IBSP/MMP13 in the presence of proinflammatory cytokines. Administration of CtBP1/2 inhibitors in fractured mice can prevent the incidence of AN. Thus, we demonstrate that the downregulation of bone healing genes dependent on proinflammatory cytokines/DNMT2/3a/CtBP1/2-p300-Runx2 axis signaling plays a critical role in the pathogenesis of AN. Disruption of this signaling may represent a new therapeutic strategy to prevent AN incidence after bone fracture.

In silico screening and identification of potential drug against p300 acetyltransferase activity in breast cancer via drug repurposing approach

Emerging evidence portray the involvement of epigenomic reprogramming in the onset and progression of several malignancies, including breast cancer. Histone acetyltransferase (HAT) p300 is a critical epigenetic regulator that acts as a transcription co-activator and regulates various cellular processes. p300 is overexpressed in breast cancer and promotes cellular invasion and survival, making it a promising druggable target. In this study, the relevance of p300 in different cancer pathways was established. Virtual screening of the FDA-approved drug library was carried out using molecular docking, and the top 10 potential repurposed drugs were identified. Further, recalculation of binding free energy of drug-p300 complexes was carried out using molecular mechanics Poisson-Boltzmann and surface area (MM-PBSA) method after molecular dynamic simulation. Based on molecular dynamic simulation parameters and binding free energy analysis, two drugs, namely Netarsudil (−305.068 kJ/mol) and Imatinib (−260.457 kJ/mol), were identified as potential repurposed drugs to inhibit the activity of p300. In conclusion, these findings suggest, Netarsudil and Imatinib might be a potential repurposed drug to combat breast cancer via p300 inhibition. Communicated by Ramaswamy H. Sarma

The RNA methyltransferase METTL16 enhances cholangiocarcinoma growth through PRDM15-mediated FGFR4 expression

BackgroundRNA N6-Methyladenosine (m6A) modification is implicated in the progression of human cancers including cholangiocarcinoma (CCA). METTL16 is recently identified as a new RNA methyltransferase responsible for m6A modification, although the role of METTL16 in CCA has not yet been examined. The current study aims to investigate the effect and mechanism of the RNA methyltransferase METTL16 in CCA.MethodsThe expression of METTL16 in CCA was examined by analyzing publicly available datasets or by IHC staining on tumor samples. siRNA or CRISPR/Cas9-mediated loss of function studies were performed in vitro and in vivo to investigate the oncogenic role of METTL16 in CCA. MeRIP-Seq was carried out to identify the downstream target of METTL16. ChIP-qPCR, immunoprecipitation, and immunoblots were used to explore the regulation mechanisms for METTL16 expression in CCA.ResultsWe observed that the expression of METTL16 was noticeably increased in human CCA tissues. Depletion of METTL16 significantly inhibited CCA cell proliferation and decreased tumor progression. PRDM15 was identified as a key target of METTL16 in CCA cells. Mechanistically, our data showed that METTL16 regulated PRDM15 protein expression via YTHDF1-dependent translation. Accordingly, we observed that restoration of PRDM15 expression could rescue the deficiency of CCA cell proliferation/colony formation induced by METTL16 depletion. Our subsequent analyses revealed that METTL16-PRDM15 signaling regulated the expression of FGFR4 in CCA cells. Specifically, we observed that PRDM15 protein was associated with the FGFR4 promoter to regulate its expression. Furthermore, we showed that the histone acetyltransferase p300 cooperated with the transcription factor YY1 to regulate METTL16 gene expression via histone H3 lysine 27 (H3K27) acetylation in CCA cells.ConclusionsThis study describes a novel METTL16-PRDM15-FGFR4 signaling axis which is crucial for CCA growth and may have important therapeutic implications. We showed that depletion of METTL16 significantly inhibited CCA cell proliferation and decreased tumor progression.

The histone acetyl transferases CBP and p300 regulate stress response pathways in synovial fibroblasts at transcriptional and functional levels

The activation of stress response pathways in synovial fibroblasts (SF) is a hallmark of rheumatoid arthritis (RA). CBP and p300 are two highly homologous histone acetyl transferases and writers of activating histone 3 lysine 27 acetylation (H3K27ac) marks. Furthermore, they serve as co-factors for transcription factors and acetylate many non-histone proteins. Here we showed that p300 but not CBP protein expression was down regulated by TNF and 4-hydroxynonenal, two factors that mimic inflammation and oxidative stress in the synovial microenvironment. We used existing RNA-sequencing data sets as a basis for a further in-depth investigation of individual functions of CBP and p300 in regulating different stress response pathways in SF. Pathway enrichment analysis pointed to a profound role of CBP and/ or p300 in regulating stress response-related gene expression, with an enrichment of pathways associated with oxidative stress, hypoxia, autophagy and proteasome function. We silenced CBP or p300, and performed confirmatory experiments on transcriptome, protein and functional levels. We have identified some overlap of CBP and p300 target genes in the oxidative stress response pathway, however, with several genes being regulated in opposite directions. The majority of stress response genes was regulated by p300, with a specific function of p300 in regulating hypoxia response genes and genes encoding proteasome subunits. Silencing of p300 suppressed proteasome enzymatic activities. CBP and p300 regulated autophagy on transcriptome and functional levels. Whereas CBP was indispensable for autophagy synthesis, silencing of p300 affected late-stage autophagy. In line with impaired autophagy and proteasome function, poly-ubiquitinated proteins accumulated after silencing of p300.

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by β-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by β-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival.

The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and the functions of specific acetylated lysine (AcK) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and nonmalignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters the regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type-dependent manner. Lysine-to-arginine substitutions indicate that although none of the AcK residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC transformed cells in vivo. The MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities.

Luteotropic and Luteolytic Factors Modulate the Expression of Nuclear Receptor Coregulators in Bovine Luteal Cells Independently of Histone Acetyltransferase and Histone Deacetylase Activities.

The aims of this study were to examine the effect of luteotropic and luteolytic factors on the mRNA and protein expression of the coactivators HAT: histone acetyltransferase p300 (P300), cyclic adenosine monophosphate response element-binding protein (CREB), and steroid receptor coactivator-1 (SRC-1) and the corepressor: nuclear receptor corepressor-2 (NCOR-2) in bovine luteal cells on days 6-10 and 16-20. HAT and HDAC activities were also measured. The obtained results showed that luteotropic and luteolytic factors influence changes in the mRNA and protein levels of the coregulators of PGRs. However, they did not affect the activity of related HAT and HDAC, respectively. Therefore, it is possible that these factors, through changes in the expression of nuclear receptor coactivators and corepressors, may affect the functioning of the nuclear receptors, including PGRs, in the bovine CL.

Characterization of molecular mechanisms driving Merkel cell polyomavirus oncogene transcription and tumorigenic potential.

Merkel cell polyomavirus (MCPyV) is associated with approximately 80% of cases of Merkel cell carcinoma (MCC), an aggressive type of skin cancer. The incidence of MCC has tripled over the past twenty years, but there are currently very few effective targeted treatments. A better understanding of the MCPyV life cycle and its oncogenic mechanisms is needed to unveil novel strategies for the prevention and treatment of MCC. MCPyV infection and oncogenesis are reliant on the expression of the early viral oncoproteins, which drive the viral life cycle and MCPyV+ MCC tumor cell growth. To date, the molecular mechanisms regulating the transcription of the MCPyV oncogenes remain largely uncharacterized. In this study, we investigated how MCPyV early transcription is regulated to support viral infection and MCC tumorigenesis. Our studies established the roles of multiple cellular factors in the control of MCPyV gene expression. Inhibitor screening experiments revealed that the histone acetyltransferases p300 and CBP positively regulate MCPyV transcription. Their regulation of viral gene expression occurs through coactivation of the transcription factor NF-κB, which binds to the viral genome to drive MCPyV oncogene expression in a manner that is tightly controlled through a negative feedback loop. Furthermore, we discovered that small molecule inhibitors specifically targeting p300/CBP histone acetyltransferase activity are effective at blocking MCPyV tumor antigen expression and MCPyV+ MCC cell proliferation. Together, our work establishes key cellular factors regulating MCPyV transcription, providing the basis for understanding the largely unknown mechanisms governing MCPyV transcription that defines its infectious host cell tropism, viral life cycle, and oncogenic potential. Our studies also identify a novel therapeutic strategy against MCPyV+ MCC through specific blockage of MCPyV oncogene expression and MCC tumor growth.

CAMP responsive element modulator α promotes effector T cells in systemic autoimmune diseases.

T lymphocytes play a crucial role in adaptive immunity. Dysregulation of T cell-derived inflammatory cytokine expression and loss of self-tolerance promote inflammation and tissue damage in several autoimmune/inflammatory diseases, including systemic lupus erythematosus (SLE) and psoriasis. The transcription factor cAMP responsive element modulator α (CREMα) plays a key role in the regulation of T cell homeostasis. Increased expression of CREMα is a hallmark of the T cell-mediated inflammatory diseases SLE and psoriasis. Notably, CREMα regulates the expression of effector molecules through trans-regulation and/or the co-recruitment of epigenetic modifiers, including DNA methyltransferases (DNMT3a), histone-methyltransferases (G9a) and histone acetyltransferases (p300). Thus, CREMα may be used as a biomarker for disease activity and/or target for future targeted therapeutic interventions.

Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis

BackgroundE1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown.MethodsCystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53.ResultsCompared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243.ConclusionThus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.

USP24-dependent stabilization of Runx2 recruits a p300/NCOA3 complex to transactivate ADAMTS genes and promote degeneration of intervertebral disc in chronic inflammation mice

BackgroundIntervertebral disc degeneration (IDD) naturally occurs during the aging process. Its occurrence is closely related to chronic inflammation; however, the causal relationship between them is controversial. This study aimed to investigate if inflammation would promote IDD incidence and explore the underlying mechanism.MethodsA chronic inflammation mouse model was established by intraperitoneal injection of lipopolysaccharide (LPS). Enzyme-linked immunosorbent assay was performed to determine proinflammatory cytokines in serum. Histological staining was used to evaluate the degeneration of IVDs. Immunoblots and RT-qPCR analyses were performed to measure protein and mRNA expression levels. Immunoprecipitation, mass spectrometry, and co-immunoprecipitation assays were used to determine the assembly of protein complex.ResultsWe found that an inflammatory microenvironment activated p38 kinase, which phosphorylated the Runx2 transcription factor at the Ser28 site. The phosphorylated Runx2 (pRunx2) then recruited a deubiquitinase, ubiquitin-specific peptidase 24 (USP24), which stabilized pRunx2 and protected it from ubiquitin-dependent proteasomal degradation. The stabilized pRunx2 recruited histone acetyltransferase p300 and nuclear receptor coactivator 3 (NCOA3) to assemble a complex. This NCOA3-p300-pRunx2 complex then transactivated the expression of 13 ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) genes, thereby promoting the degradation of extracellular matrix (ECM) in intervertebral discs (IVDs) and causing IDD. Administration of either a p38 inhibitor (doramapimod), a NCOA3 inhibitor (bufalin), or a p300 inhibitor (EML425) significantly decreased the expression of the 13 ADAMTS genes and slowed the degeneration of IVDs.ConclusionIn summary, our results demonstrate that USP24 protects pRunx2 from proteasomal degradation under chronic inflammation conditions, enabling pRunx2 to transactivate ADAMTS genes and degrade ECM. Our findings provide direct evidence that chronic inflammation triggers IDD and offer a therapeutic strategy for retarding IDD in patients with chronic inflammation.

6-Shogaol, an Active Component of Ginger, Inhibits p300 Histone Acetyltransferase Activity and Attenuates the Development of Pressure-Overload-Induced Heart Failure.

Hypertrophic stress-induced cardiac remodeling is a compensatory mechanism associated with cardiomyocyte hypertrophy and cardiac fibrosis. Continuation of this response eventually leads to heart failure. The histone acetyltransferase p300 plays an important role in the development of heart failure, and may be a target for heart failure therapy. The phenolic phytochemical 6-shogaol, a pungent component of raw ginger, has various bioactive effects; however, its effect on cardiovascular diseases has not been investigated. One micromolar of 6-shogaol suppressed phenylephrine (PE)-induced increases in cardiomyocyte hypertrophy in rat primary cultured cardiomyocytes. In rat primary cultured cardiac fibroblasts, 6-shogaol suppressed transforming growth factor-beta (TGF-β)-induced increases in L-proline incorporation. It also blocked PE- and TGF-β-induced increases in histone H3K9 acetylation in the same cells and in vitro. An in vitro p300-HAT assay revealed that 6-shogaol suppressed histone acetylation. The mice underwent transverse aortic constriction (TAC) surgery, and were administered 0.2 or 1 mg/kg of 6-shogaol daily for 8 weeks. 6-shogaol prevented TAC-induced systolic dysfunction and cardiac hypertrophy in a dose-dependent manner. Furthermore, it also significantly inhibited TAC-induced increases in histone H3K9 acetylation. These results suggest that 6-shogaol may ameliorate heart failure through a variety of mechanisms, including the inhibition of p300-HAT activity.

Accumulation of NCOA1 dependent on HERC3 deficiency transactivates matrix metallopeptidases and promotes extracellular matrix degradation in intervertebral disc degeneration.

Matrix metallopeptidases (MMPs) are critical matrix-degrading molecules and they are frequently overexpressed in degenerative discs. This study aimed to investigate the mechanism for MMP upregulation. Immunoblot and RT-qPCR were used for detecting protein and gene expression levels. 4-month-old and 24-month-old C57BL/6 mice were used for evaluating intervertebral disc degeneration (IDD). An ubiquitination assay was used to determine protein modification. Immunoprecipitation and mass spectrometry were used for identifying protein complex members. We identified the elevation of 14 MMPs among 23 members in aged mice with IDD. Eleven of these 14 MMP gene promoters contained a Runx2 (runt-related transcription factor 2) binding site. Biochemical analyses revealed that Runx2 recruited a histone acetyltransferase p300 and a coactivator NCOA1 (nuclear receptor coactivator 1) to assemble a complex, transactivating MMP expression. The deficiency of an E3 ligase called HERC3 (HECT and RLD domain containing E3 ubiquitin-protein ligase 3) resulted in the accumulation of NCOA1 in the inflammatory microenvironment. High throughput screening of small molecules that specifically target the NCOA1-p300 interaction identified a compound SMTNP-191, which showed an inhibitory effect on suppressing MMP expression and attenuating the IDD process in aged mice. Our data support a model in which deficiency of HERC3 fails to ubiquitinate NCOA1, leading to the assembly of NCOA1-p300-Runx2 and causing the transactivation of MMPs. These findings offer new insight into inflammation-mediated MMP accumulation and also provide a new therapeutic strategy to retard the IDD process.

PGE2 alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

STAT3 Inhibition Attenuates MYC Expression by Modulating Co-Activator Recruitment and Suppresses Medulloblastoma Tumor Growth by Augmenting Cisplatin Efficacy In Vivo

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.

Epigenetic Up-Regulation of ADAMTS4 in Sympathetic Ganglia is Involved in the Maintenance of Neuropathic Pain Following Nerve Injury.

Sympathetic axonal sprouting into dorsal root ganglia is a major phenomenon implicated in neuropathic pain, and sympathetic ganglia blockage may relieve some intractable chronic pain in animal pain models and clinical conditions. These suggest that sympathetic ganglia participated in the maintenance of chronic pain. However, the molecular mechanism underlying sympathetic ganglia-mediated chronic pain is not clear. Here, we found that spared nerve injury treatment upregulated the expression of ADAMTS4 and AP-2α protein and mRNA in the noradrenergic neurons of sympathetic ganglia during neuropathic pain maintenance. Knockdown the ADAMTS4 or AP-2α by injecting specific retro scAAV-TH (Tyrosine Hydroxylase)-shRNA ameliorated the mechanical allodynia induced by spared nerve injury on day 21 and 28. Furthermore, chromatin immunoprecipitation and coimmunoprecipitation assays found that spared nerve injury increased the recruitment of AP-2α to the ADAMTS4 gene promoter, the interaction between AP-2α and histone acetyltransferase p300 and the histone H4 acetylation on day 28. Finally, knockdown the AP-2α reduced the acetylation of H4 on the promoter region of ADAMTS4 gene and suppressed the increase of ADAMTS4 expression induced by spared nerve injury. Together, these results suggested that the enhanced interaction between AP-2α and p300 mediated the epigenetic upregulation of ADAMTS4 in sympathetic ganglia noradrenergic neurons, which contributed to the maintenance of spared nerve injury induced neuropathic pain.