Histone H3 Acetylation Research Articles

Phosphatidyl Ethanolamine Binding Protein FLOWERING LOCUS T-like 12 (OsFTL12) Regulates the Rice Heading Date under Different Day-Length Conditions.

Plant FLOWERING LOCUS T-Like (FTL) genes often redundantly duplicate on chromosomes and functionally diverge to modulate reproductive traits. Rice harbors thirteen FTL genes, the functions of which are still not clear, except for the Hd3a and RFT genes. Here, we identified the molecular detail of OsFTL12 in rice reproductive stage. OsFTL12 encoding protein contained PEBP domain and localized into the nucleus, which transcripts specifically expressed in the shoot and leaf blade with high abundance. Further GUS-staining results show the OsFTL12 promoter activity highly expressed in the leaf and stem. OsFTL12 knock-out concurrently exhibited early flowering phenotype under the short- and long-day conditions as compared with wild-type and over-expression plants, which independently regulates flowering without an involved Hd1/Hd3a and Ehd1/RFT pathway. Further, an AT-hook protein OsATH1 was identified to act as upstream regulator of OsFTL12, as the knock-out OsATH1 elevated the OsFTL12 expression by modifying Histone H3 acetylation abundance. According to the dissection of OsFTL12 molecular functions, our study expanded the roles intellectual function of OsFTL12 in the mediating of a rice heading date.

Trifluoromethyl quinoline derivative targets inhibiting HDAC1 for promoting the acetylation of histone in cervical cancer cells

Cervical cancer is the leading cause of death among gynecological malignant tumors, especially due to the poor prognosis of patients with advanced tumors due to recurrence, metastasis, and chemotherapy resistance. Therefore, exploring new antineoplastic drugs with high efficacy and low toxicity may bring new expectations in patients with cervical cancer. Natural products and their derivatives exert an antitumor activity. Therefore, in this work, combined with network pharmacology analysis and experimental validation, we investigated the anti-cervical cancer activity and molecular mechanism of a new trifluoromethyl quinoline (FKL) derivative in vivo and in vitro. FKL117 inhibited the proliferation of cervical cancer cells in a dose and time-dependent manner, induced apoptosis in HeLa cells, arrested the cell cycle in the G2/M phase, and regulated the expression of the apoptotic and cell cycle-related proteins Bcl-2, Bax, cyclin B1, and CDC2. We used online databases to obtain HDAC1 as one of the possible targets of FKL117 and the target binding and binding affinity were modeled by molecular docking. The results showed that FKL117 formed a hydrogen bond with HDAC1 and had good binding ability. We found that FKL117 targeted to inhibit the expression and function of HDAC1 and increased the acetylation of histone H3 and H4, which was also confirmed in vivo. The migration of HMGB1 from the nucleus to the cytoplasm further verified the above results. In conclusion, our study suggested that FKL117 might be used as a novel candidate for targeting the inhibition of HDAC1 against cervical cancer.

GLP-1 signaling-regulated SNAP25 is involved in improved insulin secretion in diabetic GK rats after Roux-en-Y gastric bypass surgery.

Roux-en-Y gastric bypass surgery (RYGB) improves glucose-stimulated insulin secretion (GSIS) in type 2 diabetes (T2D) patients. SNAP25 plays an essential role in GSIS. Clinical studies indicate that enhanced GLP-1 signaling is an important contributor to the improved β-cell function in T2D. We aimed to explore whether GLP-1-regulated SNAP25 is involved in the enhanced secretory function of β-cells in diabetic Goto-Kakizaki (GK) rats after RYGB. RYGB or sham surgery was conducted in GK rats. mRNA and protein expression of SNAP25 was assessed by qPCR and Western blot, respectively. Occupancy of CREB and acetyltransferase CBP and acetylation of histone H3 (ACH3) at the Snap25 promoter were determined using ChIP assay. RYGB led to increased SNAP25 expression and CREB phosphorylation in islets from GK rats. Increased SNAP25 improved GSIS in β-cells cultured in high glucose conditions. Consistent with increased plasma GLP-1 after RYGB, GLP-1R agonist exendin4 increased SNAP25 expression and CREB phosphorylation in β-cells. Mechanistically, exendin4 promoted the recruitment of CREB and CBP, thereby increasing ACH3 at the Snap25 promoter. Consistently, inhibition of CBP attenuated the effect of exendin4 on SNAP25 expression. Furthermore, the knockdown of SNAP25 diminished the increase of GSIS potentiated by chronic GLP-1 culture in INS-1 832/13 cells. Our findings unravel the novel mechanisms of RYGB-enhanced SNAP25 expression in β-cells, and SNAP25 may contribute to the improved β-cell secretory function induced by RYGB.

Changes in histone lysine acetylation, but not DNA methylation during facultative hibernation in Syrian hamster liver

BackgroundHibernation (torpor) is a strategy to survive extreme environmental conditions, associated with a significant decrease in metabolism and body temperature. The inducibility by the environment of torpor for facultative hibernators designates epigenetic mechanisms as likely candidates for regulation. Therefore, we set out to unravel epigenetics in the liver of a facultative hibernator, Syrian hamster (Mesocricetus auratus), sampled at different phases during hibernation, by assessing the expression of epigenetic writer and eraser enzymes, histone acetylation dynamics, and DNA methylation levels.ResultsExpression of epigenetic writers/erasers confirmed previously reported results obtained in obligatory hibernators, but might point to a mechanism specific for facultative hibernators, e.g., differential expression of histone acetyltransferases (HATs; KAT6A, KAT6B, KAT7, and KAT13D/CLOCK). These findings were in accordance with observed changes in histone H3 and H4 acetylation changes. Overall histone deacetylase (HDAC) activity was highest in torpor. No differences were detected in DNA methylation throughout all phases.ConclusionOur study thus points to histone acetylation as an important player in facultative hamster hibernation, which may underlie the orchestration of gene expression changes throughout hibernation.

Screening H3 Histone Acetylation in a Wild Bird, the House Sparrow (Passer Domesticus).

Epigenetic mechanisms are increasingly understood to have major impacts across ecology. However, one molecular epigenetic mechanism, DNA methylation, currently dominates the literature. A second mechanism, histone modification, is likely important to ecologically relevant phenotypes and thus warrants investigation, especially because molecular interplay between methylation and histone acetylation can strongly affect gene expression. There are a limited number of histone acetylation studies on non-model organisms, yet those that exist show that it can impact gene expression and phenotypic plasticity. Wild birds provide an excellent system to investigate histone acetylation, as free-living individuals must rapidly adjust to environmental change. Here, we screen histone acetylation in the house sparrow (Passer domesticus); we studied this species because DNA methylation was important in the spread of this bird globally. This species has one of the broadest geographic distributions in the world, and part of this success is related to the way that it uses methylation to regulate its gene expression. Here, we verify that a commercially available assay that was developed for mammals can be used in house sparrows. We detected high variance in histone acetylation among individuals in both liver and spleen tissue. Further, house sparrows with higher epigenetic potential in the Toll Like Receptor-4 (TLR-4) promoter (i.e., CpG content) had higher histone acetylation in liver. Also, there was a negative correlation between histone acetylation in spleen and TLR-4 expression. In addition to validating a method for measuring histone acetylation in wild songbirds, this study also shows that histone acetylation is related to epigenetic potential and gene expression, adding a new study option for ecological epigenetics.

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Shared Mechanisms in Pparγ1sv and Pparγ2 Expression in 3T3-L1 Cells: Studies on Epigenetic and Positive Feedback Regulation of Pparγ during Adipogenesis.

We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator-activated receptor-γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5' untranslated region (5'UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (-733 to -76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR-Cas9 system identified crucial PPARγ-binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3-L1 cells.

Extracellular cold-inducible RNA-binding protein mediated neuroinflammation and neuronal apoptosis after traumatic brain injury.

Extracellular cold-inducible RNA-binding protein (eCIRP) plays a vital role in the inflammatory response during cerebral ischaemia. However, the potential role and regulatory mechanism of eCIRP in traumatic brain injury (TBI) remain unclear. Here, we explored the effect of eCIRP on the development of TBI using a neural-specific CIRP knockout (KO) mouse model to determine the contribution of eCIRP to TBI-induced neuronal injury and to discover novel therapeutic targets for TBI. TBI animal models were generated in mice using the fluid percussion injury method. Microglia or neuron lines were subjected to different drug interventions. Histological and functional changes were observed by immunofluorescence and neurobehavioural testing. Apoptosis was examined by a TdT-mediated dUTP nick end labelling assay in vivo or by an annexin-V assay in vitro. Ultrastructural alterations in the cells were examined via electron microscopy. Tissue acetylation alterations were identified by non-labelled quantitative acetylation via proteomics. Protein or mRNA expression in cells and tissues was determined by western blot analysis or real-time quantitative polymerase chain reaction. The levels of inflammatory cytokines and mediators in the serum and supernatants were measured via enzyme-linked immunoassay. There were closely positive correlations between eCIRP and inflammatory mediators, and between eCIRP and TBI markers in human and mouse serum. Neural-specific eCIRP KO decreased hemispheric volume loss and neuronal apoptosis and alleviated glial cell activation and neurological function damage after TBI. In contrast, eCIRP treatment resulted in endoplasmic reticulum disruption and ER stress (ERS)-related death of neurons and enhanced inflammatory mediators by glial cells. Mechanistically, we noted that eCIRP-induced neural apoptosis was associated with the activation of the protein kinase RNA-like ER kinase-activating transcription factor 4 (ATF4)-C/EBP homologous protein signalling pathway, and that eCIRP-induced microglial inflammation was associated with histone H3 acetylation and the α7 nicotinic acetylcholine receptor. These results suggest that TBI obviously enhances the secretion of eCIRP, thereby resulting in neural damage and inflammation in TBI. eCIRP may be a biomarker of TBI that can mediate the apoptosis of neuronal cells through the ERS apoptotic pathway and regulate the inflammatory response of microglia via histone modification.

Immunostaining for Epigenetic Modifications in Fagopyrum Calli.

Immunostaining is a well-established technique for identifying specific proteins in tissue samples with specific antibodies to identify a single target protein. It is commonly used in research and provides information about cellular localization and protein expression levels. This chapter describes a detailed protocol for immunostaining fixed Fagopyrum calli embedded in Steedman's wax using nine antibodies raised against histone H3 and H4 methylation and acetylation on several lysines and DNA methylation.

Quantitative Analysis of Epigenetic Modifications in Fagopyrum Nuclei with Confocal Microscope, ImageJ, and R Studio.

Epigenetic programming plays a vital role in regulating pluripotency genes, which become activated or inactivated during the processes of dedifferentiation and differentiation during an organism's development. The analysis of epigenetic modifications has become possible through the technique of immunostaining, where specific antibodies allow the identification of a single target protein. This chapter describes a detailed protocol for the analysis of the epigenetic modifications with the use of confocal microscopy, subsequent image, and statistical analysis on the example of Fagopyrum calli with the use of nine antibodies raised against histone H3 and H4 methylation and acetylation on several lysines as well as DNA methylation.

Focal adhesion kinase and its epigenetic interactors as diagnostic and therapeutic hints for pediatric hepatoblastoma.

Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification and therapies remains a focal point to improve the outcome for high-risk patients. Here, we pointed to explore the impact of these mechanisms in HB. An observational study was performed on liver samples from a cohort of 17 patients with a diagnosis of HB and two normal liver samples. The in vitro experiments were executed on the Huh6 human HB cell line treated with the FAK inhibitor TAE226. Our results highlight a significant up-regulation of mRNA and protein expression of FAK in livers from HB with respect to normal livers. The increased protein expression of total and Tyr397 phosphorylated FAK (pTyr397FAK) was significantly correlated with the expression of some epigenetic regulators of histone H3 methylation and acetylation. Of note, the expression of pTyr397FAK, N-methyltransferase enzyme (EZH2) and tri-methylation of the H3K27 residue correlated with tumor size and alpha-fetoprotein (AFP) levels. Finally, TAE226 caused a significant reduction of pTyr397FAK, epigenetic regulators, AFP, EPCAM, OCT4, and SOX2, in association with anti-proliferative and pro-apoptotic effects on HB cells. Our results suggest a role of FAK in HB that requires further investigations mainly focused on the exploration of its effective diagnostic and therapeutic translatability.

The Tudor-knot Domain of KAT5 Regulates Nucleosomal Substrate Acetylation

The lysine acetyltransferase KAT5 is a pivotal enzyme responsible for catalyzing histone H4 acetylation in cells. In addition to its indispensable HAT domain, KAT5 also encompasses a conserved Tudor-knot domain at its N-terminus. However, the function of this domain remains elusive, with conflicting findings regarding its role as a histone reader. In our study, we have employed a CRISPR tiling array approach and unveiled the Tudor-knot motif as an essential domain for cell survival. The Tudor-knot domain does not bind to histone tails and is not required for KAT5’s chromatin occupancy. However, its absence leads to a global reduction in histone acetylation, accompanied with genome-wide alterations in gene expression that consequently result in diminished cell viability. Mechanistically, we find that the Tudor-knot domain regulates KAT5’s HAT activity on nucleosomes by fine-tuning substrate accessibility. In summary, our study uncovers the Tudor-knot motif as an essential domain for cell survival and reveals its critical role in modulating KAT5’s catalytic efficiency on nucleosome and KAT5-dependent transcriptional programs critical for cell viability.

Chokeberry reduces inflammation in human preadipocytes

Chokeberry, Aronia melanocarpa, is an indigenous fruit from North America used as food and to prevent chronic disease by Indigenous Peoples. The objective of this study was to test anti-inflammatory effects of anthocyanin on palmitic acid (PA)-induced IL-6 gene expression, IL-6 DNA methylation, and histone (H3) acetylation. Additionally, we examined effects of anthocyanins Cyanidin-3-O-galactoside (C3Gal) and Cyanidin-3-glucoside (C3G) on IL-6 gene expression. Human primary pre-adipocytes were treated with chokeberry juice extract (CBE), C3Gal or C3G in the presence or absence of PA or lipopolysaccharide (LPS). CBE inhibited LPS- and PA-induced IL-6 mRNA expression (p < 0.0001), while C3G and C3Gal had smaller effects. Human IL-6 promoter DNA methylation was increased (p = 0.0256) in CBE treated cells compared to control. Histone H3 acetylations were not affected by CBE or PA treatment. These data indicate that CBE epigenetically reduced PA-induced inflammation by regulating IL-6 DNA methylation without affecting histone modifications in human preadipocyte cells.

HDAC1-3 inhibition increases SARS-CoV-2 replication and productive infection in lung mesothelial and epithelial cells.

Despite the significant progress achieved in understanding the pathology and clinical management of SARS-CoV-2 infection, still pathogenic and clinical issues need to be clarified. Treatment with modulators of epigenetic targets, i.e., epidrugs, is a current therapeutic option in several cancers and could represent an approach in the therapy of viral diseases. Aim of this study was the analysis of the role of histone deacetylase (HDAC) inhibition in the modulation of SARS-CoV-2 infection of mesothelial cells (MCs).MeT5A cells, a pleura MC line, were pre-treated with different specific class I and IIb HDAC inhibitors. Unexpectedly, treatment with HDAC1-3 inhibitors significantly increased ACE2/TMPRSS2 expression, suggesting a role in favoring SARS-CoV-2 infection. We focused our analysis on the most potent ACE2/TMPRSS2 inducer among the inhibitors analysed, MS-275, a HDAC1-3 inhibitor. ACE2/TMPRSS2 expression was validated by Western Blot (WB) and immunofluorescence. The involvement of HDAC inhibition in receptor induction was confirmed by HDAC1/HDAC2 silencing. In accordance to the ACE2/TMPRSS2 expression data, MS-275 increased SARS-CoV-2 replication and virus propagation in Vero E6 cells.Notably, MS-275 was able to increase ACE2/TMPRSS2 expression and SARS-CoV-2 production, although to a lesser extent, also in the lung adenocarcinoma cell line Calu-3 cells.Mechanistically, treatment with MS-275 increased H3 and H4 histone acetylation at ACE2/TMPRSS2 promoters, increasing their transcription. This study highlights a previously unrecognized effect of HDAC1-3 inhibition in increasing SARS-CoV-2 cell entry, replication and productive infection correlating with increased expression of ACE2 and TMPRSS2. These data, while adding basic insight into COVID-19 pathogenesis, warn for the use of HDAC inhibitors in SARS-CoV-2 patients.

Histone H3 Acetylation Is Involved in Retinoid Acid-Induced Neural Differentiation through Increasing Mitochondrial Function.

Histone acetylation and mitochondrial function contribute importantly to neural differentiation, which is critically associated with neurodevelopmental disorders such as Down Syndrome (DS). However, whether and how histone acetylation regulates mitochondrial function and further affects neural differentiation has not been well described. In this study, when treated with retinoid acid (RA), the human neuroblastoma SH-SY5Y cell line was used as a neural differentiation model. We found that the acetylation of histone H3, especially H3 lysine 14 acetylation (H3K14ac), and mitochondrial function, including biogenesis and electron transport chain, were enhanced during neural differentiation. Specific inhibition of histone acetyltransferases (HATs) induced neural differentiation deficits, accompanied by downregulation of mitochondrial function. Furthermore, RA receptors (RARs) interacting with HATs were involved in the increased H3K14ac and the enhanced mitochondrial function during the neural differentiation process. Finally, receptor-interacting protein 140 (RIP140), a co-repressor of RARs, was also involved in regulating histone acetylation. RIP140 overexpression inhibited histone acetylation and mediated negative feedback on target genes which are involved in RA signaling. These findings evidenced that when interacting with RARs which had been negatively regulated by RIP140, RA promoted neural differentiation by promoting H3K14ac and enhanced mitochondrial function. This provides a molecular foundation for further investigations into abnormal neural development.

MHY446 induces apoptosis via reactive oxygen species-mediated endoplasmic reticulum stress in HCT116 human colorectal cancer cells

This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.

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.

Abstract A044: The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer

Abstract KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A044.

Chromosome-specific maturation of the epigenome in the Drosophila male germline.

Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.