Promoter Sites Research Articles

The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

Role of the Egr2 Promoter Antisense RNA in Modulating the Schwann Cell Chromatin Landscape

Background: Schwann cells (SCs) and their plasticity contribute to the peripheral nervous system’s capacity for nerve regeneration after injury. The Egr2/Krox20 promoter antisense RNA (Egr2-AS) recruits chromatin remodeling complexes to inhibit Egr2 transcription following peripheral nerve injury. Methods: RNA-seq and ATAC-seq were performed on control cells, Lenti-GFP-transduced cells, and cells overexpressing Egr2-AS (Lenti-AS). Egr2 AS-RNA was cloned into the pLVX-DsRed-Express2-N1 lentiviral expression vector (Clontech, Mountain View, CA, USA), and the levels of AS-RNA expression were determined. Ezh2 and Wdr5 were immunoprecipitated from rat SCs and RT-qPCR was performed against AS-Egr2 RNA. ChIP followed by DNA purification columns was used to perform qPCR for relevant promoters. Hi-C, HiC-DC+, R, Bioconductor, and TOBIAS were used for significant and differential loop analysis, identifications of COREs and CORE-promotor loops, comparisons of TF activity at promoter sites, and identification of site-specific TF footprints. OnTAD was used to detect TADs, and Juicer was used to identify A/B compartments. Results: Here we show that a Neuregulin-ErbB2/3 signaling axis mediates binding of the Egr2-AS to YY1Ser184 and regulates its expression. Egr2-AS modulates the chromatin accessibility of Schwann cells and interacts with two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables targeting of H3K27me3 and H3K4me3 to promoters of Egr2 and C-JUN, respectively. Expression of the Egr2-AS results in reorganization of the global chromatin landscape and quantitative changes in the loop formation and contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the Egr2-AS induces changes in the hierarchical TADs and increases transcription factor binding scores on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Conclusions: Our results show that Neuregulin-ErbB2/3-YY1 regulates the expression of Egr2-AS, which mediates remodeling of the chromatin landscape in Schwann cells.

Abstract 4134539: Hybrid Cardiomyocyte Targeting Peptide Down-regulates NF-κB Pathway in Human Cardiomyocytes

Introduction: Role of NF-κB driven inflammation leading to heart failure with preserved ejection fraction has received little attention. Our previous in vivo work with cardiac targeting peptide (CTP) in guinea pigs identified suppression of NF- κB pathway by CTP. In the current work we sought to further study the effects of a hybrid version of CTP (hCTP: amino acids at positions 3 and 11 substituted with D-amino acids) on TNF-α mediated NF-κB activation, as well as expression of its downstream inflammatory markers IL-1β and IL-8. Methods: Human cardiomyocytes (CMCs) were transfected using Lipofectamine 3000 with a reporter luciferase plasmid carrying an NF-κB promoter site upstream of the luciferase gene. Cells were treated with various concentrations of hCTP for 24 hours, followed by a TNF-α challenge (20ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to above manipulations was assessed by FACS using live-dead stain. CMCs were treated as above, and cell culture supernatants analyzed for IL-8 levels using human ELISA kit. In another set of experiments, CMCs were transfected with NF-κB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, incubated with 10nM angiotensin 2 (Ang2) and 100nM phenylephrine (PE) for 24hrs to assess levels of IL-1β in the cell culture media using an ELISA assay. Results: hCTP inhibited TNF-α mediated NF-κB activation with a dose-response curve, with maximum inhibition with hCTP seen at 10µM concentrations (p<0.001). Treatment with hCTP inhibited the TNF-α induced IL-8 secretion with significant decreases seen even at the 1µM dose. Similarly, secretion of IL-1β production by CMCs in response to Ang2/PE stimulation was suppressed by hCTP even at the lowest tested dose (1µM; p<0.5). Conclusions: Our data suggests that hCTP inhibited NF-κB pathway transcriptional activity in response to TNF-α stimulation in a dose-dependent fashion in human CMCs. Furthermore, treatment with hCTP inhibited TNF-α induced IL-8 and IL-1β secretion even at doses as low as 1µM. The ability of hCTP to efficiently targeting this inflammatory pathway suggests that it has therapeutic potential in the treatment of heart failure. Further in vivo studies are ongoing to test this hypothesis.

BIOM-07. SURVIVAL-ASSOCIATED GENETIC VARIATIONS IN TAIWANESE GLIOBLASTOMA MULTIFORME PATIENTS - A RETROSPECTIVE NGS STUDY

Abstract This retrospective study, conducted at Taichung Veterans General Hospital in Taiwan, employed Next-Generation Sequencing (NGS) technology to analyze genetic variations associated with survival in patients with glioblastoma multiforme (GBM). The study encompassed 30 newly diagnosed GBM patients, divided into two groups based on survival duration: long-term (over two years) and short-term (under two years). The findings are outlined below: Key Mutations: Significant mutations included CHEK2, IDH1, and TP53, along with variations in the TERT promoter and TP53 RNA splicing sites. Correlation Between Mutations and Survival: Contrary to Western data, TP53 mutations were more prevalent in the long-term survival group, suggesting that regional genetic differences may influence GBM prognosis. Therapeutic Effectiveness: The application of bevacizumab was significantly associated with improved survival rates, indicating its potential efficacy in treatment. The study highlights the critical role of personalized medicine in the treatment of GBM in Taiwan and points to the necessity of further research into the genetic characteristics and treatment responses of GBM patients in Taiwan and potentially other non-Western regions. These insights underline the importance of continuing to explore effective personalized treatment strategies and enhancing our understanding of genetic determinants in diverse populations. Moreover, the prevalence of certain mutations in Taiwanese patients, which differ from those commonly observed in Western populations, prompts a reevaluation of global treatment frameworks and suggests the potential for region-specific therapeutic protocols. The implication of these findings extends beyond immediate clinical applications, providing a foundation for future clinical trials and research into genetic markers that could dictate treatment choices. In conclusion, this study not only underscores the importance of individualized treatment plans based on genetic profiling but also highlights the variations in genetic mutations across different populations, which could have profound implications for the management of GBM worldwide. Further investigation into these disparities and their implications on treatment efficacy is essential for advancing GBM treatment and improving patient outcomes globally.

Peroxisome proliferator-activated receptor gamma regulates interleukin-6 induced lipoprotein(a) gene expression in human HepG2 cells

Abstract Background Lipoprotein(a)[Lp(a)] is an independent risk factor for atherosclerotic cardiovascular disease (ASCVD) and outcomes. The LPA gene promoter contains an interleukin 6 (IL-6) responsive binding site. IL-6 is a pro-inflammatory cytokine and data from patients with inflammatory diseases indicate an impact of the inflammatory milieu on LPA gene regulation and Lp(a) production. In a clinical study, the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone was associated with decreased Lp(a) levels. Publicly accessible ChIP-sequencing of human hepatocytes and measures of Lp(a) production by HepG2 cells provide an opportunity to explore PPARγ regulation of hepatic Lp(a) production. Purpose To investigate the role of PPARγ as a regulator of hepatic Lp(a) production. Methods Public ChIP-sequencing of human hepatocytes was analyzed. Commercially available HepG2 cells were incubated with one of the following: (1) scrambled small interfering RNA (siRNA) alone (control); (2) IL-6 (20ng/mL) and scrambled siRNA ; (3) IL-6 and PPARγ siRNA ; and (4) IL-6 and pioglitazone (25uM). LPA transcription was measured using the quantitative polymerase chain reaction. Results Analysis of the ChIP-sequencing data demonstrated that the LPA locus variant rs56393506, which is associated with increased Lp(a) levels and ASCVD risk in the general population, was within the location of the PPARγ binding site in the LPA promoter (Figure 1). IL-6 significantly increased LPA transcription in the HepG2 cells, compared to control (1.65±0.44 vs. 0.66±0.45, p=0.0027). In the presence of siRNA-mediated knock-down of PPARγ (58±17% PPARγ reduction, p<0.0001 vs. scrambled control), the effect of IL-6 on LPA transcription was significantly amplified (6.78±2.4 vs. 1.65±0.44, p=0.0009). IL-6 did not increase LPA transcription in the presence of the PPARγ agonist pioglitazone compared to control (0.16±0.03 vs. 0.66+0.45, p=0.11, Figure 2). Conclusion PPARγ is a negative regulator of IL-6 induced hepatic Lp(a) production and may represent a new therapeutic target in patients with inflammatory conditions.Figure 1Figure 2

Novel Fosfomycin Resistance Mechanism in Pseudomonas entomophila Due to Atypical Pho Regulon Control of GlpT

Background/Objectives: Pseudomonas entomophila is a ubiquitous bacterium capable of killing insects of different orders and has become a model for host–pathogen studies and a promising tool for biological pest control. In the human pathogen Pseudomonas aeruginosa, spontaneous resistance to fosfomycin arises almost exclusively from mutations in the glycerol-3-phosphate transporter (GlpT), the drug’s sole entry route in this species. Here, we investigated whether this specificity is conserved in P. entomophila, as it could provide a valuable marker system for studying mutation rates and spectra and for selection in genetic engineering. Methods: We isolated 16 independent spontaneous fosfomycin-resistant mutants in P. entomophila, and studied the genetic basis of the resistance using a combination of sequencing, phenotyping and computational approaches. Results: We only found two mutants without alterations in glpT or any of its known regulatory elements. Whole-genome sequencing revealed unique inactivating mutations in phoU, a key regulator of the phosphate starvation (Pho) regulon. Computational analyses identified a PhoB binding site in the glpT promoter, and experiments showed that phoU inactivation reduced glpT expression nearly 20-fold. While placing a sugar-phosphate transporter under the Pho regulon may seem advantageous, bioinformatic analysis shows this configuration is atypical among pseudomonads. Conclusions: this atypical Pho regulon control of GlpT probably reflects the peculiarities of P. entomophila’s habitat and lifestyle; highlighting how readily regulatory evolution can lead to the rapid divergence of resistance mechanisms, even among closely related species.

Structural insights into the DNA-binding mechanism of BCL11A: The integral role of ZnF6.

The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α2γ2) to adult hemoglobin (HbA: α2β2) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a different role compared to ZnF4 and 5, providing a positive entropic contribution to DNA binding and γ-globin gene repression. Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and β-thalassemia.

Mechanisms underlying TRPV4-mediated regulation of miR-146a expression.

Persistent inflammation is a major contributor in the development of various inflammatory diseases like atherosclerosis. Our study investigates how transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, interacts with microRNA-146a (miR-146a), within the context of inflammation and atherosclerosis. Micro-RNAs play a critical role in controlling gene expression, and miR-146a is notable for its anti-inflammatory actions. TRPV4 is activated by diverse soluble and mechanical stimuli, and often associated with inflammatory responses in various diseases. Here, we find that TRPV4 negatively regulates miR-146a expression in macrophages, especially following stimulation by lipopolysaccharides or alterations in matrix stiffness. We show that in atherosclerosis, a condition characterized by matrix stiffening, TRPV4 decreases miR-146a expression in aortic tissue macrophages. We find that TRPV4's impact on miR-146a is independent of activation of NFκB, Stat1, P38, and AKT, but is rather mediated through a mechanism involving histone deacetylation instead of DNA methylation at the miR-146a promoter site. Furthermore, we show that N-terminal residues 1 to 130 in TRPV4 is essential in suppression of miR-146a expression in LPS-stimulated macrophages. Altogether, this study identifies a regulatory mechanism of miR-146a expression by TRPV4 which may open new potential therapeutic strategies for managing inflammatory diseases.

Long Terminal Repeats of Gammaretroviruses Retain Stable Expression after Integration Retargeting.

Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations.

Palmitic acid causes hepatocyte inflammation by suppressing the BMAL1-NAD+-SIRT2 axis.

Palmitic acid is the most abundant saturated fatty acid in circulation and causes hepatocyte toxicity and inflammation. As saturated fatty acid can also disrupt the circadian rhythm, the present work evaluated the connection between clock genes and NAD+ dependent Sirtuins in protecting hepatocytes from lipid-induced damage. Hepatocytes (immortal cells PH5CH8, hepatoma cells HepG2) treated with higher doses of palmitic acid (400-600μM) showed typical features of steatosis accompanied with growth inhibition and increased level of inflammatory markers (IL-6 IL-8, IL-1α and IL-1β) together with decline in NAD+ levels. Palmitic acid treated hepatocytes showed significant decline in not only the protein levels of SIRT2 but also its activity as revealed by the acetylation status of its downstream targets (Tubulin and NF-ƙB). Additionally, the circadian expression of both SIRT2 and BMAL1 was inhibited in presence of palmitic acid in only the non-cancerous hepatocytes, PH5CH8 cells. Clinical specimens obtained from subjects with NASH-associated fibrosis, ranging from absent (F0) to cirrhosis (F4), showed a significant decline in levels of SIRT2 and BMAL1, especially in the cirrhotic liver. Ectopic expression of BMAL1 or activating SIRT2 by supplementation with nicotinamide riboside (precursor of NAD+) dampened the palmitic acid induced lipoinflammation and lipotoxicity more effectively in PH5CH8 cells as compared to HepG2 cells. Mechanistically, palmitic acid caused transcriptional suppression of SIRT2 by disrupting the chromatin occupancy of BMAL1 at its promoter site. Overall, the work suggested that SIRT2 is a clock-controlled gene that is transcriptionally regulated by BMAL1. In conclusion the activation of the BMAL1-NAD+-SIRT2 axis shows hepatoprotective effects by preventing lipotoxicity and dampening inflammation.

CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.

The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We firstperformed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences forenhancingthe heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.

DNA methylation changes in the genome of patients with hypogonadotropic hypogonadism

Although some Mendelian neurodevelopmental disorders have been shown to entail specific DNA methylation changes designated as epi-signatures, it remains unknown whether epi-signatures are consistent features of other genetic disorders. Here, we analyzed DNA methylation profiles of patients with hypogonadotropic hypogonadism (HH), a rare neuroendocrine disorder typically caused by monogenic or oligogenic mutations. First, we performed microarray-based genome-wide methylation analyses of nine patients with HH due to ANOS1, SOX2, or SOX10 variants and 12 control individuals. The results showed that 1118 probes were differentially methylated in one or more patients. The differentially methylated probes were highly variable among patients. No significant methylation changes were observed in genes functionally associated with ANOS1, SOX2, or SOX10. Then, we performed pyrosequencing of six selected CpG sites in the nine patients and 35 additional HH patients. The results of the patients were compared with those of 48 fertile men. There were no common methylation changes among these patients, with the exception of hypermethylation of two CpG sites in the ZNF245 promoter of three patients. Hypermethylation of the promoter has previously been reported as a very rare epigenetic polymorphism in the general population. These results indicate that genomes of HH patients have considerable DNA methylation changes; however, these changes are more likely to be physiological epigenetic variations than disease-specific epi-signatures. Our data suggest a possible association between hypermethylation of the ZNF254 promoter and HH, which needs to be examined in future studies.

Abstract P364: Chromatin Interactions Between Regulatory Elements Influence Gene Expression in Human iPSC-Derived Endothelial and Vascular Smooth Muscle Cells

Most SNPs associated with blood pressure (BP) are located in noncoding regions of the human genome, where they may regulate gene expression in BP-relevant cell types. However, the mechanisms by which these SNPs influence BP regulation are unclear. To address this, we developed comprehensive epigenomic landscapes in human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) and vascular smooth muscle cells (iVSMCs), incorporating DNA methylation analysis (RRBS), transcriptome analysis (RNA-seq), chromatin state profiling (ATAC-seq), and chromatin interaction mapping (Micro-C). We identified 5301, 8916, and 6046 chromatin loops in iECs and 5329, 7598, and 5228 loops in iVSMCs at 4, 8, and 16 kb resolutions (FDR < 0.05) respectively based on the Micro-C analysis. Remarkably, 82.4% and 83.8% of these chromatin contact regions in iECs and iVSMCs, respectively, harbored at least one known regulatory element such as an enhancer, promoter, or transcription factor binding site. Notably, 109 and 119 BP-associated SNPs were found within the chromatin contact regions of iECs and iVSMCs, respectively. We further investigated chromatin contact regions that involved interactions of regulatory elements. In both iECs and iVSMCs, lower methylation levels were observed in chromatin contact regions involving promoter-promoter (PP) and enhancer-promoter (EP) interactions, compared to enhancer-enhancer (EE), enhancer-transcription factor binding site (ET), and promoter-transcription factor binding site (PT) interactions. Additionally, greater chromatin accessibility was observed in chromatin contact regions involving PP and EP interactions. BP-associated SNPs were significantly enriched in chromatin contact regions involving PP and EP interactions. Moreover, genes proximal to chromatin contact regions involving PP or EP interactions exhibited higher expression than those near EE or other regulatory element interactions. These findings offer new insights into how chromatin interactions between regulatory elements define transcriptional states in human endothelial and vascular smooth muscle cells, providing a novel basis for understanding the genetic regulation of BP.

Zeta class glutathione S-transferase is involved in phoxim tolerance and is potentially regulated by the transcription factor CncC in Agrotis ipsilon (Lepidoptera: Noctuidae)

The black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae), is an important agricultural pest. Phoxim is an organophosphate insecticide that has been widely used to control A. ipsilon. The extensive application of phoxim has resulted in a reduction in phoxim susceptibility in A. ipsilon. However, the molecular mechanisms underlying phoxim tolerance in A. ipsilon remain unclear. In this work, we report the involvement of AiGSTz1, a zeta class glutathione S-transferase, in phoxim tolerance in A. ipsilon. Exposure to a sublethal concentration (LC50) of phoxim dramatically upregulated the transcription level of the AiGSTz1 gene in A. ipsilon larvae, and this upregulation might be caused by phoxim-induced oxidative stress. The recombinant AiGSTz1 protein expressed in Escherichia coli was able to metabolize phoxim. Furthermore, AiGSTz1 displayed antioxidant activity to protect against oxidative stress. Knockdown of AiGSTz1 by RNA interference significantly increased the mortality rate of A. ipsilon larvae in response to phoxim. In addition, the transcription factor AiCncC can bind to the cap ‘n’ collar isoform C: muscle aponeurosis fibromatosis (CncC:Maf) binding site in the putative promoter of the AiGSTz1 gene. Silencing of AiCncC resulted in a dramatic downregulation of AiGSTz1. These results indicated that AiGSTz1 is involved in phoxim tolerance and is potentially regulated by AiCncC. These findings provide valuable insights into the defense mechanisms used by A. ipsilon against phoxim.

Saikosaponin A Recovers Impaired Filaggrin Levels in Inflamed Skin by Downregulating the Expression of FRA1 and c-Jun.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter-reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter-reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the -343/+25 FLG promoter-reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

DNA Methylation Effects on Van der Woude Syndrome Phenotypic Variability.

Van der Woude Syndrome (VWS) presents with combinations of lip pits (LP) and cleft lip and/or cleft palate (CL/P, CPO). VWS phenotypic heterogeneity even amongst relatives, suggests that epigenetic factors may act as modifiers. IRF6, causal for 70% of VWS cases, and TP63 interact in a regulatory loop coordinating epithelial proliferation and differentiation in palatogenesis. We hypothesize that differential DNA methylation within IRF6 and TP63 regulatory regions underlie VWS phenotypic discordance. DNA methylation of CpG sites in IRF6 and TP63 promoters and in an IRF6 enhancer element was compared amongst blood or saliva DNA samples of 78 unrelated cases. Analyses were done separately for blood and saliva, within each sex and in combination, and to address cleft type (CL/P ± LP vs. CPO ± LP) and phenotypic severity (any cleft + LP vs. any cleft only). For cleft type, blood samples showed higher IRF6 and TP63 promoter methylation on males with CPO ± LP compared to CL/P ± LP and on individuals with CPO ± LP compared to those with CL/P ± LP, respectively. Saliva samples showed higher IRF6 enhancer methylation on individuals with CPO ± LP compared to CL/P ± LP and contrary to above, lower TP63 promoter methylation on CPO ± LP compared to CL/P ± LP. For phenotypic severity, blood samples showed no differences; however, saliva samples showed higher IRF6 promoter methylation in individuals with any cleft + LP compared to those without lip pits. We observed differential methylation in IRF6 and TP63 regulatory regions associated with cleft type and phenotypic severity, indicating that epigenetic changes in IRF6 and TP63 can contribute to phenotypic heterogeneity in VWS.

KLF4 Induces Colorectal Cancer by Promoting EMT via STAT3 Activation.

Krüppel-like factor 4 (KLF4) has been demonstrated to exert a pro-carcinogenic effect in solid tissues. However, the precise biological function and underlying mechanisms in colorectal cancer (CRC) remains elucidated. To investigate whether KLF4 participates in the proliferation and invasion of CRC. The expression of KLF4 was investigated using immunohistochemistry and immunoblotting. The clinical significance of KLF4 was evaluated. Furthermore, the effect of inhibiting or overexpressing KLF4 on tumor was examined. Immunoblotting and qPCR were used to detect Epithelial-mesenchymal transition-related proteins levels. Additionally, the molecular function of KLF4 is related to the STAT3 signaling pathway and was determined through JASPAR, GSEA analysis, and in vitro experiments. KLF4 exhibits down-regulated expression in CRC and is part of the vessel invasion, TNM stage, and worse prognosis. In vitro studies have shown that KLF4 promotes cellular proliferation and invasion, as well as EMT processes. Xenograft tumor models confirmed the oncogenic role of KLF4 in nude mice. Furthermore, GSEA and JASPAR databases analysis reveal that the binding of KLF4 to the signal transducer and activator of transcription 3 (STAT3) promoter site induces activation of p-STAT3 signaling. Subsequent targeting of STAT3 confirmed its pivotal role in mediating the oncogenic effects exerted by KLF4. The study suggests that KLF4 activates STAT3 signaling, inducing epithelial-mesenchymal transition, thereby promoting CRC progression.

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Nrf2/FSP1/CoQ10 axis-mediated ferroptosis is involved in sodium aescinate-induced nephrotoxicity

Sodium aescinate (SA), an active compound found in horse chestnut seeds, is widely used in clinical practice. Recently, the incidence of SA-induced adverse events, particularly renal impairment, has increased. Our previous work demonstrated that SA causes severe nephrotoxicity via nephrocyte ferroptosis; however, the underlying mechanism remains to be fully elucidated. In the current study, we investigated additional molecular pathways involved in SA-induced nephrotoxicity. Our results showed that SA inhibited cell viability, disrupted cellular membrane integrity, and enhanced reactive oxygen species (ROS), ferrous iron (Fe2+), and malondialdehyde (MDA) levels, as well as lipid peroxidation in rat proximal renal tubular epithelial cell line (NRK-52E) cells. SA also depleted coenzyme Q10 (CoQ10, ubiquinone) and nicotinamide adenine dinucleotide (NADH) and reduced ferroptosis suppressor protein 1 (FSP1) and polyprenyltransferase (coenzyme Q2, COQ2) activity, triggering lipid peroxidation and ROS accumulation in mouse kidneys and NRK-52E cells. The overexpression of COQ2, FSP1, or CoQ10 (ubiquinone) supplementation effectively attenuated SA-induced ferroptosis, whereas iFSP1 or 4-formylbenzoic acid (4-CBA) pretreatment exacerbated SA-induced nephrotoxicity. Additionally, SA decreased nuclear factor-erythroid-2-related factor 2 (Nrf2) levels and inhibited Nrf2 binding to the −1170/-1180 bp ARE site in FSP1 promoter, resulting in FSP1 suppression. Overexpression of Nrf2 or its agonist dimethyl fumarate (DMF) promoted FSP1 expression, thereby improving cellular antioxidant capacity and alleviating SA-induced ferroptosis. These results suggest that SA-triggers renal injury through oxidative stress and ferroptosis, driven by the suppression of the Nrf2/FSP1/CoQ10 axis.

Upstream CtrA-binding sites both induce and repress pilin gene expression in Caulobacter crescentus

Pili are bacterial surface structures important for surface adhesion. In the alphaproteobacterium Caulobacter crescentus, the global regulator CtrA activates transcription of roughly 100 genes, including pilA which codes for the pilin monomer that makes up the pilus filament. While most CtrA-activated promoters have a single CtrA-binding site at the − 35 position and are induced at the early to mid-predivisional cell stage, the pilA promoter has 3 additional upstream CtrA-binding sites and it is induced at the late predivisional cell stage. Reporter constructs where these additional sites were disrupted by deletion or mutation led to increased activity compared to the WT promoter. In synchronized cultures, these mutations caused pilA transcription to occur approximately 20 min earlier than WT. The results suggested that the site overlapping the − 35 position drives pilA gene expression while the other upstream CtrA-binding sites serve to reduce and delay expression. EMSA experiments showed that the − 35 Site has lower affinity for CtrA∼P compared to the other sites, suggesting binding site affinity may be involved in the delay mechanism. Mutating the upstream inhibitory CtrA-binding sites in the pilA promoter caused significantly higher numbers of pre-divisional cells to express pili, and phage survival assays showed this strain to be significantly more sensitive to pilitropic phage. These results suggest that pilA regulation evolved in C. crescentus to provide an ecological advantage within the context of phage infection.