Increased Histone H3 Acetylation Research Articles

Decursin Inhibits Expression and Nuclear Localization of DNMT and HDAC, and Suppresses the Growth and Survival of Prostate Cancer Cells

Background: DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors have utility in cancer therapeutics. However, they are associated with toxicity, which limits their efficacy against carcinomas. Previously, we reported that decursin has anticancer activity against prostate cancer (PCa). In this study, we investigated its epigenetic role in inhibiting the survival of PCa cells. Methods and Results: PCa cell viability was evaluated using MTT and trypan blue assays. The effect of decursin on global methylation and gene-specific methylation was assessed by dot blot assay and MS-PCR, respectively. Differential expression of DNMTs and HDACs was analyzed through semi-quantitative RT-PCR and western blotting. The activity of these enzymes was measured using commercial kits. The nuclear localization of DNMTs and HDAC3 was assessed via immunoblotting. Results: Decursin decreased cell viability and global methylation in PCa cells. It diminished the expression of DNMT1, DNMT3A, DNMT3B, and pan-DNMT activities. Decursin also reduced nuclear levels and increased cytosolic levels of DNMT1 and DNMT3A. Additionally, it inhibited the expression and activity of HDACs, leading to increased total acetylation and H3K9/14 acetylation of histone H3. A reduction in nuclear localization of HDAC3 was observed in decursin-treated PCa cells. Overall, decursin reduced DNMT and HDAC expression and enhanced the nuclear export of DNMT1, DNMT3A, and HDAC3, leading to decreased activities of these enzymes. Conclusion: This study demonstrates the potent anticancer effects of decursin in prostate cancer cells through modulation of epigenetic mechanisms. Decursin decreased both overall and nuclear levels of DNMTs and HDACs. This effect is likely due to decursin’s inhibitory action on these enzymes, as indicated by decreased global methylation and increased histone H3 acetylation. These findings suggest that decursin has multi-faceted epigenetic modulatory properties, making it a promising therapeutic agent for prostate cancer prevention.

Manipulating mitochondrial pyruvate carrier function causes metabolic remodeling in corneal myofibroblasts that ameliorates fibrosis

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.

FBP1 orchestrates keratinocyte proliferation/differentiation and suppresses psoriasis through metabolic control of histone acetylation

Keratinocyte proliferation and differentiation in epidermis are well-controlled and essential for reacting to stimuli such as ultraviolet light. Imbalance between proliferation and differentiation is a characteristic feature of major human skin diseases such as psoriasis and squamous cell carcinoma. However, the effect of keratinocyte metabolism on proliferation and differentiation remains largely elusive. We show here that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) promotes differentiation while inhibits proliferation of keratinocyte and suppresses psoriasis development. FBP1 is identified among the most upregulated genes induced by UVB using transcriptome sequencing and is elevated especially in upper epidermis. Fbp1 heterozygous mice exhibit aberrant epidermis phenotypes with local hyperplasia and dedifferentiation. Loss of FBP1 promotes proliferation and inhibits differentiation of keratinocytes in vitro. Mechanistically, FBP1 loss facilitates glycolysis-mediated acetyl-CoA production, which increases histone H3 acetylation at lysine 9, resulting in enhanced transcription of proliferation genes. We further find that the expression of FBP1 is dramatically reduced in human psoriatic lesions and in skin of mouse imiquimod psoriasis model. Fbp1 deficiency in mice facilitates psoriasis-like skin lesions development through glycolysis and acetyl-CoA production. Collectively, our findings reveal a previously unrecognized role of FBP1 in epidermal homeostasis and provide evidence for FBP1 as a metabolic psoriasis suppressor.

The hydroxamic acid derivative YPX-C-05 alleviates hypertension and vascular dysfunction through the PI3K/Akt/eNOS pathway

Hypertension is a prevalent cardiovascular disease characterized by elevated blood pressure and increased vascular resistance. HDAC inhibitors have emerged as potential therapeutic agents due to their ability to modulate gene expression and cellular processes. YPX-C-05, a novel hydroxamic acid-based HDAC inhibitor, shows promise in its vasodilatory effects and potential targets for hypertension treatment. In this study, we aimed to elucidate the mechanisms underlying YPX-C-05's vasodilatory effects and explore its therapeutic potential in hypertension. To determine the ex vivo vasodilatory effects of YPX-C-05, isolated aortic rings precontracted with phenylephrine were used. We assessed YPX-C-05's inhibitory effects on HDACs and its impact on histone H4 deacetylation levels in endothelial cells. Network pharmacology analysis was employed to predict putative targets of YPX-C-05 for hypertension treatment. To investigate the involvement of the PI3K/Akt/eNOS pathway, we employed enzyme-linked immunosorbent assay and to assess the levels of NO, ET-1, BH2, and BH4 in human umbilical vein endothelial cells. And we also analyzed the mRNA expression of eNOS and ET-1. Furthermore, Western blotting was conducted to quantify the phosphorylated and total Akt and eNOS levels in human umbilical vein endothelial cell lysates following treatment with YPX-C-05. In order to elucidate the vasodilatory mechanism of YPX-C-05, we employed pharmacological inhibitors for evaluation purposes. Furthermore, we evaluated the chronic antihypertensive effects of YPX-C-05 on N-omega-nitro-L-arginine-induced hypertensive mice in an in vivo model. Vascular remodeling was assessed through histological analysis. Our findings demonstrated that YPX-C-05 exerts significant vasodilatory effects in isolated aortic rings precontracted with phenylephrine. Furthermore, YPX-C-05 exhibited inhibitory effects on HDACs and increased histone H4 acetylation in endothelial cells. Network pharmacology analysis predicted YPX-C-05 might activate endothelial eNOS via PI3K/Akt signaling pathway. Inhibition of the PI3K/Akt/eNOS pathway attenuated the vasodilatory effects of YPX-C-05, as evidenced by reduced levels of phosphorylated Akt and eNOS in human umbilical vein endothelial cell lysates. The chronic administration of YPX-C-05 in N-omega-nitro-L-arginine-induced hypertensive mice resulted in significant antihypertensive effects. Histological analysis demonstrated a reduction in vascular remodeling, further supporting the therapeutic potential of YPX-C-05 in hypertension. This study demonstrates for the first time that the novel hydroxamic acid-based HDAC inhibitor YPX-C-05 produces significant antihypertensive and vasodilatory effects through the PI3K/Akt/eNOS pathway. Our findings support the developing prospect of YPX-C-05 as a novel antihypertensive drug.

BAP18 acting as a novel peroxisome proliferator-activated receptor α co-regulator contributes to hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is a common malignancy worldwide with a poor prognosis. The therapeutic outcomes of HCC patients are urgently needed to be improved, and predictive biomarkers for the optimal treatment selection remains to be further defined. In the present study, our results showed that BPTF-associated protein of 18 KDa (BAP18) was highly expressed in HCC tissues. In cultured HCC cells, BAP18 regulated a subset of down-stream genes involved in different functions, particularly including peroxisome proliferator-activated receptor (PPAR) pathway and lipid metabolism. Furthermore, BAP18 co-activated PPARα-mediated transactivation and facilitated the recruitment of nucleosome acetyltransferase of H4 (NuA4)/tat interacting protein 60 (TIP60) complex, thereby increasing histone H4 acetylation on stearoyl-CoA desaturase 1 (SCD1) loci. In addition, BAP18 promoted HCC cell proliferation, increased intracellular lipid levels and enhanced cell survival under the metabolic stress conditions, such as glucose limitation or tyrosine kinase inhibitors (TKIs) treatment. Importantly, higher BAP18 expression was positively correlated with the postoperative recurrence and the poor disease-free survival in clinical patients receiving sorafenib treatment. Altogether, we discovered that BAP18 plays an oncogenic role in the survival and proliferation of HCC cells, and BAP18 may serve as a predictive biomarker for adjunct TKIs treatment in patients with HCC, and further facilitate the precise treatment.

Oxidized ATM governs stemness of breast cancer stem cell through regulating ubiquitylation and acetylation switch

Cancer stem cells (CSCs), as parts of tumor initiation cells, play a crucial role to tumorigenesis, development and recurrence. However, the complicated mechanisms of CSCs to adapt to tumor microenvironment and its stemness maintenance remains unclear. Here, we show that oxidized ATM, a hypoxia-activated cytoplasm ATM, acts a novel function to maintain CSC stemness in triple-negative breast cancer cells (BCSCs) via regulating histone H4 acetylation. Mechanistically, oxidized ATM phosphorylates TRIM21 (a E3 ubiquitin ligase) serine 80 and serine 469. Serine 80 phosphorylation of TRIM21 is essential for the ubiquitination activity of TRIM21. TRIM21 binds with SIRT1 (one of deacetylase), resulting in ubiquitylation-mediated degradation of SIRT1. The reduced SIRT1 leads to increase of histone H4 acetylation, thus facilitating CSC-related gene expression. Clinical data verify that high level of ATM in breast tumors is positively correlated with malignant grade, and is closely related with low SIRT1, high p-TRIM21, and high CD44 expression. In conclusion, our study provides a novel mechanism by which oxidized ATM governing BCSCs stemness and reveals an important link among oxidized ATM, histone acetylation, and BCSCs maintenance.

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

Trps1 acts as a regulator of Sf-1 transcription and testosterone synthesis in mouse Leydig cells.

Infertility has attracted global concern, and disruption of testosterone is a common cause of male infertility. Exploring the critical factors in testosterone biosynthesis may provide new insights for disease research and clinical therapy. Research on trichorhinophalangeal syndrome-1 (Trps1) gene has recently been focus on cancers; it is yet unknown whether Trps1 produces a marked effect in the male reproductive system. In the current study, single-cell RNA sequencing analysis of trichorhinophalangeal syndrome-1 gene (Trps1) expression in mouse testes and cleavage under targets and tagmentation and RNA sequencing were utilized to investigate the functionality of Trps1 in mouse Leydig cells. Knockdown of Trps1 increased testosterone synthesis in vitro and vivo using adeno-associated viral delivery and conditional knockout models. The results showed that Trps1 was abundantly expressed in Leydig cells. The expression levels of both steroidogenic factor-1 (Sf-1) and steroidogenic enzymes (Cyp11a1, Hsd3b, Cyp17a1, and Hsd17b3) as well as testosterone secretion were increased after Trps1 deficiency in vivo and vitro. Furthermore, disruption of Trps1 reduced histone deacetylase 1/2 activity and increased histone H3 acetylation in the Sf-1 promoter, thereby promoting testosterone secretion. Interestingly, Sf-1 also regulated the transcription of Trps1 through activating transcription factor 2. These results indicate that Trps1 targets Sf-1 to affect steroidogenesis through histone acetylation and shed light on the critical role of Trps1 functioning in the mouse Leydig cells.

Inhibition of ACSS2 attenuates alcoholic liver steatosis via epigenetically regulating de novo lipogenesis.

Steatosis is the early pathological change in alcohol-associated liver disease. However, its precise mechanism is still unclear. The present study is aimed to explore the role and mechanism of acetyl-CoA synthetase 2 (ACSS2) in acute alcohol-induced lipogenesis. The increase in ACSS2 nuclear import and histone H3 acetylation were observed in mice after intraperitoneally injected with 2 g/kg ethanol or oral administration of 5 g/kg ethanol and also validated in hepatocytes stimulated with ethanol or acetate. The role of ACSS2 was further explored in liver-specific ACSS2 knockdown mice fed with ethanol-containing diet. Alcohol challenge induced hepatic lipid deposition and upregulated lipogenic genes in mice. It also promoted ACSS2 nuclear import and increased histone H3 acetylation. In hepatocytes, ethanol induced similar phenomena whereas ACSS2 knockdown blocked histone acetylation and lipogenic gene induction. P300/CBP associated factor (PCAF), but not general control nonderepressible 5, CREB-binding protein (CBP) and p300, facilitated H3K9 acetylation responding to ethanol challenge. CUT&RUN assay showed the enrichment of acetylated histone H3K9 surrounding Fasn and Acaca promoters. These results indicated that ethanol metabolism promoted ACSS2 nuclear import to support lipogenesis via H3K9 acetylation. In alcohol-feeding mice, liver-specific ACSS2 knockdown blocked the interaction between PCAF and H3K9 and suppressed lipogenic gene induction in the liver, demonstrating the critical role of ACSS2 in lipogenesis. Our study demonstrated that alcohol metabolism generated acetyl-CoA in the nucleus dependently on nuclear ACSS2, contributing to epigenetic regulation of lipogenesis in hepatic steatosis. Targeting ACSS2 may be a potential therapeutical strategy for acute alcoholic liver steatosis.

DNA damage, demethylation and anticancer activity of DNA methyltransferase (DNMT) inhibitors

Role of DNA damage and demethylation on anticancer activity of DNA methyltransferase inhibitors (DNMTi) remains undefined. We report the effects of DNMT1 gene deletion/disruption (DNMT1−/−) on anticancer activity of a class of DNMTi in vitro, in vivo and in human cancers. The gene deletion markedly attenuated cytotoxicity and growth inhibition mediated by decitabine, azacitidine and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) in colon and breast cancer cells. The drugs induced DNA damage that concurred with DNMT1 inhibition, subsequent G2/M cell-cycle arrest and apoptosis, and upregulated p21 in DNMT1+/+ versus DNMT1−/− status, with aza-T-dCyd the most potent. Tumor growth and DNMT1 were significantly inhibited, and p21 was upmodulated in mice bearing HCT116 DNMT1+/+ xenograft and bladder PDX tumors. DNMT1 gene deletion occurred in ~ 9% human colon cancers and other cancer types at varying degrees. Decitabine and azacitidine demethylated CDKN2A/CDKN2B genes in DNMT1+/+ and DNMT1−/− conditions and increased histone-H3 acetylation with re-expression of p16INK4A/p15INK4B in DNMT1−/− state. Thus, DNMT1 deletion confers resistance to DNMTi, and their anti-cancer activity is determined by DNA damage effects. Patients with DNMT1 gene deletions may not respond to DNMTi treatment.

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Increased histone H3 acetylation inhibit the inflammatory response and activate the serum immunity of Pearl oyster Pinctada fucata martensii

To produce cultured pearls, a mantle graft with a nucleus is transplanted into a host pearl oyster, this process is called “transplantation”. The immune response of pearl oyster after transplantation is a major factor that leads to nucleus rejection and death. Butyrate is a histone deacetylase (HDAC) inhibitor which can inhibit the deacetylation process of histones and effectively reduce the inflammatory response. To clarify the function of histone acetylation in immune response after transplantation, butyrate (10 mmol/L) was used for the treatment of pearl oysters before transplantation. Results showed that the proportion of histone H3 acetylation of the hemocytes was significantly increased after butyrate treatment before transplantation (BH group) compared with the control group at 6–24 h. Transcriptome analysis showed that butyrate treatment activated the “lysosome”, inhibited cell migration and cell proliferation at 6 and 12 h, respectively, and activated the intracellular immune recognition response of pearl oyster at 24 h after transplantation. The apoptosis detection revealed no significant difference in the proportion of apoptotic cells between the control and BH group. Moreover, butyrate treatment increased the activity of some immune-related enzymes in the serum of pearl oyster after transplantation.

An enhanced network of energy metabolism, lysine acetylation, and growth-promoting protein accumulation is associated with heterosis in elite hybrid rice

Heterosis refers to the superior performance of a hybrid compared with its parental lines. Although several genetic and molecular models have been proposed to explain heterosis, it remains unclear how hybrid cells integrate complementary gene expression or activity to drive heterotic growth. In this work, we show that accumulation of growth-promoting and energy metabolism proteins, enhanced energy metabolism activities, and increased protein lysine acetylation were associated with superior growth of the panicle meristem in the elite hybrid rice Shanyou 63 relative to its parental varieties. Metabolism of nuclear/cytosolic acetyl-coenzyme A was also enhanced in the hybrid, which paralleled increases in histone H3 acetylation to selectively target the expression of growth-promoting and metabolic genes. Lysine acetylation of cellular proteins, including TARGET OF RAPAMYCIN complex 1, ribosomal proteins, and energy metabolism enzymes, was also augmented and/or remodeled to modulate their activities. The data indicate that an enhanced network of energy-producing metabolic activity and growth-promoting histone acetylation/gene expression in the hybrid could contribute to its superior growth rate and may constitute a model to explain heterosis.

Dietary ketone body-escalated histone acetylation in megakaryocytes alleviates chemotherapy-induced thrombocytopenia.

Chemotherapy-induced thrombocytopenia (CIT) is a severe complication in patients with cancer that can lead to impaired therapeutic outcome and survival. Clinically, therapeutic options for CIT are limited by severe adverse effects and high economic burdens. Here, we demonstrate that ketogenic diets alleviate CIT in both animals and humans without causing thrombocytosis. Mechanistically, ketogenic diet-induced circulating β-hydroxybutyrate (β-OHB) increased histone H3 acetylation in bone marrow megakaryocytes. Gain- and loss-of-function experiments revealed a distinct role of 3-β-hydroxybutyrate dehydrogenase (BDH)-mediated ketone body metabolism in promoting histone acetylation, which promoted the transcription of platelet biogenesis genes and induced thrombocytopoiesis. Genetic depletion of the megakaryocyte-specific ketone body transporter monocarboxylate transporter 1 (MCT1) or pharmacological targeting of MCT1 blocked β-OHB-induced thrombocytopoiesis in mice. A ketogenesis-promoting dietalleviated CIT in mouse models. Moreover, a ketogenic diet modestly increased platelet counts without causing thrombocytosis in healthy volunteers, and a ketogenic lifestyle inversely correlated with CIT in patients with cancer. Together, we provide mechanistic insights into a ketone body-MCT1-BDH-histone acetylation-platelet biogenesis axis in megakaryocytes and propose a nontoxic, low-cost dietary intervention for combating CIT.

LncRNA IL21-AS1 interacts with hnRNPU protein to promote IL21 overexpression and aberrant differentiation of Tfh cells in systemic lupus erythematosus.

The aberrant differentiation of T follicular helper (Tfh) cells plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanism of regulating Tfh cells differentiation remains unclear. Long noncoding RNAs (lncRNAs) act as important regulators in the processes of innate and adaptive immune response. Whether lncRNAs are involved in regulating Tfh cell differentiation and autoimmune responses need to be further identified. The characters and functions of human IL21-AS1 and its mouse homologous lncRNA (mIl21-AS) were investigated by a series of biochemical assays and cell transfection assay. mIl21-AS1 regulating humoral immune response in vivo was explored by keyhole limpet haemocyanin (KLH) and chronic graft versus host disease (cGVHD) model. Human IL21-AS1 and its mouse homologous lncRNA (mIl21-AS) were identified and cloned. We uncovered that IL21-AS1 was highly expressed in CD4+ T cells of SLE patients and Tfh cells, which promoted differentiation of Tfh cells. Mechanistically, IL21-AS1 bound heterogeneous nuclear ribonucleoprotein U and recruited acetyltransferases CREB-binding protein to the promoter of IL21, leading to the transcriptional activation of IL21 and Tfh cells differentiation through increasing Histone H3 acetylation level on IL21 promoter. Moreover, Tfh proportion and antibodies production were significantly increased in mIl21-AS knock-in mice immunized with KLH. mIl21-AS1 overexpression also exacerbated the lupus-like phenotype in cGVHD mice model. Our results demonstrate that IL21-AS1 activates IL21 transcription via epigenetic mechanism to promote germinal centre response, adding insight into the molecular regulation of autoimmune pathogenesis and providing a novel target for SLE treatment.

Abstract 15331: Epigenetic Interaction of Grk4 and Slc4a5 Variants Regulates Gene Transcription and Blood Pressure

Salt sensitive hypertension is associated with variants of G protein-coupled receptor kinase 4γ (GRK4), as well as with activating variants of the solute carrier family 4 member 5 (rs7571842 and rs10177833; SLC4a5), which encodes the sodium bicarbonate transporter 2 (NBCe2). We tested the hypothesis that the effect of the R65L variant (rs2960306) of GRK4 (GRK4γ 65L) on salt-sensitivity involves a gene-gene interaction with SLC4a5 variants. We found that the renal expression of SLC4a5 is increased in salt sensitive transgenic mice expressing GRK4γ 65L and is higher (50%; n=4; P<0.02) than that in GRK4 γ expressing the wild type variant (WT). Moreover, that AAV9-mediated renal specific overexpression of SLC4a5 in GRK4γ 65L mice increases the blood pressure (BP) response to a high salt diet compared with mice expressing empty vector (n=3; P<0.05). We studied human renal proximal tubule cells (hRPTCs) endogenously expressing GRK4 WT and SLC4a5 WT, GRK4 65L, SLC4a5 variants and both GRK4 65L and SLC4a5 variants. SLC4A5 expression is increased in hRPTCs expressing GRK4 65L and in cells expressing both GRK4 65L and SLC4a5 variants (n=5, P<0.05) compared with GRK4 WT. GRK4 can interact with nuclear histone deacetylases (HDACs) and co-immunoprecipitates with HDAC1 in nuclear fractions of hRPTCs. Renal-selective downregulation of HDAC1 increases BP in C57Bl/6J (P<0.05) mice. hRPTCs carrying both GRK4 65L and SLC4a5 variants have decreased expression (60%; n=4; P<0.05) of HDAC1 compared with cells expressing GRK4 WT and SLC4a5 WT or GRK4 65L This correlates with increased histone H4 acetylation but unchanged H2A, H2B and H3 acetylation in cells carrying both variants. Our results indicate a complex interaction between these variants in the regulation not only of SLC4a5 expression but that of other genes involved in salt sensitivity.

ODP571 Blockade of ErbB-2 Nuclear Function Induces the Interferon Signaling Pathway in Breast Cancer Models Resistant to Trastuzumab

Abstract ErbB-2, a member of ErbB family of receptor tyrosine kinases, is a key oncogenic driver in breast cancer. Despite clinical efficiency of ErbB-2-targeted therapies (trastuzumab, TZ), resistance to drugs is a major issue in the clinic. While ErbB-2 is mainly a plasma membrane-bound receptor, it also migrates to the nucleus (NErbB-2) where it can act as a transcription factor or coactivator. We previously reported that NErbB-2 is a major proliferation driver in TZ-resistant breast cancer. To investigate the NErbB-2 dependent transcriptome, RNAseq was performed using a TZ-resistant breast cancer model (JIMT-1 cells) with high constitutive levels of NErbB-2. JIMT-1 cells were transfected with an ErbB-2 nuclear localization domain mutant (hErbB-2ΔNLS), which also acts as a dominant-negative inhibitor of endogenous NErbB-2 migration. Exclusion of ErbB-2 from the nucleus resulted in up-regulation of 280 genes and down-regulation of 33 genes. Functional analysis revealed that NErbB-2 blockade enriched the expression of genes involved in type-I interferon (IFN) signaling pathway. IFNB1 and its downstream effectors OAS2 and TRIM22 were among the top up-regulated genes. In an independent breast cancer model (i. e., HCC-1569 cells), exclusion of NErbB-2 from the nucleus also induced expression of these genes. Blockade of NErbB-2 localization by injection of the hErbB-2ΔNLS mutant into JIMT-1 tumor xenografts significantly inhibited in vivo tumor growth and induced mRNA expression of IFNB1, OAS2 and TRIM22. Interestingly, blockade of NErbB-2 localization by treatment with Retro-2, an inhibitor of the retrograde transport, showed similar effects consistent with modulation of the IFN signaling pathway by NErbB-2. Bioinformatic analyses showed that both the promoter and the coding region of the IFNB1 gene contain ErbB-2 associated sequences (HAS sites). ChIP-PCR analyses revealed ErbB-2 recruitment to the HAS sites of the IFNB1 promoter and coding regions in normal growth conditions. Transfection of JIMT-1 cells with the hErbB-2ΔNLS mutant abolished the recruitment of ErbB-2 at the IFNB1 gene and also caused an increase in histone H4 acetylation, a marker of active gene transcription. NErbB-2 immunostaining in a cohort of 32 primary invasive ErbB-2-positive breast carcinomas treated with TZ revealed that NErbB-2 expression correlated with a poor disease-free survival. While this cohort is small, the findings suggest that NErbB-2 could be used as a biomarker of poor response to TZ in the clinic. In summary, our findings indicate that NErbB-2 drives the growth of TZ-resistant breast cancer cells via transcriptional repression of the IFNB1 signaling pathway, and highlight NErbB-2 as a therapeutic target and biomarker in TZ-resistant breast cancer. Presentation: No date and time listed

The chromatin remodeler BRAHMA recruits HISTONE DEACETYLASE6 to regulate root growth inhibition in response to phosphate starvation in Arabidopsis.

Plasticity in root system architecture (RSA) allows plants to adapt to changing nutritional status in the soil. Phosphorus availability is a major determinant of crop yield, and RSA remodeling is critical to increasing the efficiency of phosphorus acquisition. Although substantial progress has been made in understanding the signaling mechanism driving phosphate starvation responses in plants, whether and how epigenetic regulatory mechanisms contribute is poorly understood. Here, we report that the Switch defective/sucrose non-fermentable (SWI/SNF) ATPase BRAHMA (BRM) is involved in the local response to phosphate (Pi) starvation. The loss of BRM function induces iron (Fe) accumulation through increased LOW PHOSPHATE ROOT1 (LPR1) and LPR2 expression, reducing primary root length under Pi deficiency. We also demonstrate that BRM recruits the histone deacetylase (HDA) complex HDA6-HDC1 to facilitate histone H3 deacetylation at LPR loci, thereby negatively regulating local Pi deficiency responses. BRM is degraded under Pi deficiency conditions through the 26 S proteasome pathway, leading to increased histone H3 acetylation at the LPR loci. Collectively, our data suggest that the chromatin remodeler BRM, in concert with HDA6, negatively regulates Fe-dependent local Pi starvation responses by transcriptionally repressing the RSA-related genes LPR1 and LPR2 in Arabidopsis thaliana.

AIMP1 promotes multiple myeloma malignancy through interacting with ANP32A to mediate histone H3 acetylation

Multiple myeloma (MM) is the second most common hematological malignancy. An overwhelming majority of patients with MM progress to serious osteolytic bone disease. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) participates in several steps during cancer development and osteoclast differentiation. This study aimed to explore its role in MM. The gene expression profiling cohorts of MM were applied to determine the expression of AIMP1 and its association with MM patient prognosis. Enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting were used to detect AIMP1 expression. Protein chip analysis, RNA-sequencing, and chromatin immunoprecipitation and next-generation sequencing were employed to screen the interacting proteins and key downstream targets of AIMP1. The impact of AIMP1 on cellular proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro and a xenograft model in vivo. Bone lesions were evaluated using tartrate-resistant acid phosphatase staining in vitro. A NOD/SCID-TIBIA mouse model was used to evaluate the effect of siAIMP1-loaded exosomes on bone lesion formation in vivo. AIMP1 expression was increased in MM patients and strongly associated with unfavorable outcomes. Increased AIMP1 expression promoted MM cell proliferation in vitro and in vivo via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Protein chip assays and subsequent experiments revealed that AIMP1 interacted with acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) to regulate histone H3 acetylation. In addition, AIMP1 increased histone H3 acetylation enrichment function of GRB2-associated and regulator of MAPK protein 2 (GAREM2) to increase the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2). Furthermore, AIMP1 promoted osteoclast differentiation by activating nuclear factor of activated T cells c1 (NFATc1) in vitro. In contrast, exosome-coated small interfering RNA of AIMP1 effectively suppressed MM progression and osteoclast differentiation in vitro and in vivo. Our data demonstrate that AIMP1 is a novel regulator of histone H3 acetylation interacting with ANP32A in MM, which accelerates MM malignancy via activation of the MAPK signaling pathway.