Promoter Sites Research Articles

CRISPR/Cas9 Driven Targeted Editing of Grain Number 1a gene: sgRNA Constructs Design for Yield Enhancement in Rice (Oryza sativa L.)

Yield is the ultimate trait determined by various quantitative trait loci (QTLs). One such QTL, GRAIN NUMBER 1a (GN1a), encodes for an enzyme Cytokinin oxidase/ dehydrogenase (CKX), which negatively influences the yield by degrading the phytohormone Cytokinin. CRISPR/Cas9, a precise means for targeted editing of a gene for the improvement of a particular trait in plants. Therefore, targeted genome editing of GN1a gene was performed to down-regulate the expression using CRISPR/Cas9 for the increase in grain number and yield with improved Cytokinin content in the panicle meristem. For the site-targeted mutagenesis, the single guide RNAs (sgRNAs) were designed using plant specific CRISPR-P v2.0 software. Two efficient sgRNAs were chosen critically entrenched on their GC content, on-target values, location on the gene, off-target sites and their location, secondary structures, adjacent to the Protospacer Adjacent Motif (PAM) NGG. The binary vector pRGEB32, with Cas9 influenced by rice ubiquitin promoter and BsaI restriction site driven by rice U3 promoter was employed for cloning of sgRNAs. The sgRNAs were tempered, phosphorylated and astringed with the binary vector pRGEB32, transformed into E. coli DH5α initially, then mobilized into A. tumefaciens EHA105. The present study helps in the development of elite lines which will lead to enhancement of grain number and overall yield for the growing population and farmer’s welfare.

A KSHV RNA-binding protein promotes FOS to inhibit nuclease AEN and transactivate RGS2 for AKT phosphorylation.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an RNA-binding protein ORF57 in lytic infection. Using an optimized CLIP-seq in this report, we identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS RNA as one of the major ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5' end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is likely competitive to the binding of host nuclease AEN (ISG20L1) of which physiological RNase activity remains unknown. KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits AEN transcription through FOS binding to AEN promoter but transactivates RGS2, a regulator of G-protein-coupled receptors. FOS binds a conserved AP-1 site in the RGS2 promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection.IMPORTANCEWe discovered that FOS, a heterodimer component of oncogenic transcription factor AP-1, is highly elevated in KSHV-infected cells by expression of a viral lytic RNA-binding protein, ORF57, which binds a 13-nt RNA motif near the FOS RNA 5' end to prolong FOS RNA half-life. This binding of ORF57 to FOS RNA is competitive to the binding of host RNA destabilizer(s). KSHV infection inhibits expression of host nuclease AEN, but not MTR4. FOS inhibits AEN transcription by binding to the AEN promoter but transactivates RGS2 by binding to a conserved AP-1 site in the RGS2 promoter, thereby enhancing RGS2 expression and phosphorylation of AKT. Thus, KSHV lytic infection controls the expression of a subset of genes for signaling, cell cycle progression, and proliferation to potentially contribute to viral oncogenesis.

Long-range regulation of transcription scales with genomic distance in a gene-specific manner.

Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.

A catalog of gene editing sites and genetic variations in editing sites in model organisms

BackgroundCRISPR-Cas systems require a protospacer adjacent motif (PAM), which plays an essential role in self/non-self discrimination in their natural context, to cleave DNA for genome editing. Unfortunately, common genetic variation is distributed throughout genomes, which can block recognition of target sites by Cas proteins. However, little information is available about the distribution of editing sites in model organisms and how often common variation overlaps with those PAM sites.ResultsHerein, we characterized six representative Cas proteins (Cas9, Cas12a, Cas12b, Cas12i, Cas12j and Cas12l) genomic editing sites in ten model organisms (yeast, flatworms, flies, zebrafish, mice, humans, rice, maize, Arabidopsis and tomato). We demonstrated that there were more than 34 editing sites per kilobase on average in these genomes. In each genome, 91.69–99.83% and 95.4–99.73% of genes had at least one unique editing site in exon and promoter, respectively. Depending on publicly available genomic diversity data, we identified the variations (SNPs and InDels) in editing sites in humans and rice, indicating the risk in the application of CRISPR/Cas technology. Finally, using CCR5 and BCL11A as examples, we revealed variation site was a factor that must be considered when designing sgRNA.ConclusionsOur findings not only revealed the distribution characteristics of editing sites of six representative Cas proteins in ten model organism genomes but also shed light on the adverse effect of variation sites on target site recognition. Our current work will serve as a reminder of the risks of CRISPR application.

The miR172a-SNB module orchestrates induced and adult resistance to multiple diseases via MYB30-mediated lignin accumulation in rice

Plants mount induced resistance and adult plant resistance against different pathogens during the whole growth period. Rice production faces threats from multiple major diseases, including rice blast, sheath blight, and bacterial leaf blight. Here we report that a microRNA module, miR172a-SNB-MYB30, regulates induced resistance and adult plant resistance to these three major diseases via lignification in rice. Mechanistically, pathogens induce expression of miR172a, which down-regulates transcription factor SNB to release its suppression on MYB30, leading to an increase in lignin biosynthesis and disease resistance during the whole growth period. Moreover, expression levels of miR172a and MYB30 are gradually increased and consistently correlated with lignin contents and disease resistance during rice development reaching a peak at full maturity stage, whereas SNB RNA levels are reversely correlated with lignin contents and disease resistance, indicating the involvement of the miR172a-SNB-MYB30 module in plant adult resistance. A high conservation exists in the functional domain of SNB protein and its binding sites in the MYB30 promoter among over 4000 rice accessions; moreover, abnormal expression of miR172a, SNB, or MYB30 compromises yield traits, suggesting an artificial selection of the miR172a-SNB-MYB30 module during rice domestication. Our results reveal a novel role for a conserved miRNA-regulated module that significantly contributes to induced and adult resistance against multiple pathogens via lignin accumulation, advancing our understanding in broad-spectrum resistance and adult resistance.

Transcriptome-wide investigation and functional characterization reveal a terpene synthase involved in γ-terpinene biosynthesis in Monarda citriodora.

Monarda citriodora Cerv. ex Lag. is a rich source of industrially important compounds like γ-terpinene, carvacrol, thymol and thymoquinone. Understanding the regulation of γ-terpinene biosynthesis, a precursor for other monoterpenes, could facilitate upscaling of these metabolites in M. citriodora. Therefore, the present study aimed to unravel and characterize the terpene synthase (TPS) involved in γ-terpinene biosynthesis. Homology searches revealed 33 TPS members in the transcriptome assembly of M. citriodora. Based on the correlation of expression patterns and phytochemical profile, McTPS22 emerged as the putative TPS for γ-terpinene biosynthesis. Molecular docking suggested geranyl diphosphate (GPP) as a potential substrate for McTPS22. Heterologous expression in Escherichia coli and Nicotiana benthamiana confirmed the role of McTPS22 in γ-terpinene biosynthesis. Both in-silico prediction and confocal microscopy indicated plastidial localization of the McTPS22. Gene co-expression network analysis revealed 507 genes interacting with McTPS22, including 80 transcription factors (TFs). Of these, 46 TFs had binding sites in the McTPS22 promoter, and 36 showed significant correlations with γ-terpinene accumulation, suggesting they may be potential regulators. Promoter analysis indicated regulation by phytohormones and abiotic factors, confirmed by phytohormone elicitation and QRT-PCR. The histochemical GUS staining suggested that McTPS22 is primarily active in the glandular trichomes of M. citriodora. The present work provides insights into the molecular regulation of biosynthesis of γ-terpinene in M. citriodora.

Social Media Practices of Small and Medium Enterprises in Masbate Province: Impact on Industry Competitiveness

Small and medium enterprises (SMEs) can benefit significantly from social media due to its user-friendly nature, cost-effectiveness, and widespread accessibility. By leveraging social media platforms, SMEs can effectively communicate with customers, market their products, facilitate internal collaboration, and provide customer sup-port at minimal costs.This research assessed the use of social media among SMEs in the fashion, food, beverages, cosmetics, and clothing industries in Masba-te province to enhance competitiveness. It focused on key areas such as customer engagement, social network marketing, website promo-tion, customer relationship management, and brand management, while also evaluating industry competitiveness based on customer satisfaction, loyalty, operational efficiency, and profitability.A quantitative descriptive approach was employed to evaluate variables and their relationships. Employees from SMEs were ran-domly selected as respondents, while industry customers were cho-sen through purposive sampling. A self-designed Likert-scale survey captured respondents' perspectives on the impact of social media practices on industry competitiveness, combining both quantitative and qualitative insights.The analysis included descriptive statistics, such as percentages and frequencies, to outline respondent profiles, and means and stand-ard deviations to examine trends in social media practices among SMEs. Pearson correlation analysis was conducted to assess the effec-tiveness of these practices, while a t-test was used to highlight differ-ences in perceptions between respondent groups regarding social media effectiveness and industry competitiveness.

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

CGGBP1 is a GC-rich DNA-binding protein which is important for genomic integrity, gene expression and epigenome maintenance through regulation of CTCF occupancy and cytosine methylation. It has remained unclear how CGGBP1 integrates multiple diverse functions with its simple architecture of only a DNA-binding domain tethered to a C-terminal tail with low structural rigidity. We have used truncated forms of CGGBP1 with or without the DNA-binding domain (DBD) to assay cytosine methylation and global gene expression. Proximal promoters of CGGBP1-repressed genes, although significantly GC-poor, contain GC-rich transcription factor binding motifs and exhibit base compositions indicative of low C-T transition rates due to prevention of cytosine methylation. Genome-wide analyses of cytosine methylation and binding of CGGBP1 DBD show that CGGBP1 restricts cytosine methylation in a manner that depends on its DBD and its DNA-binding. The CGGBP1-repressed genes show an increase in promoter cytosine methylation alongside a decrease in transcript abundance when the DBD-deficient CGGBP1 is expressed. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss and thereby regulates gene expression. By analysing orthologous promoter sequences we show that restriction of cytosine methylation is a function of CGGBP1 progressively acquired during vertebrate evolution. A superimposition of our results and evolution of CGGBP1 suggests that mitigation of cytosine methylation is majorly achieved by its N-terminal DBD. Our results position CGGBP1 DNA-binding as a major evolutionarily acquired mechanism through which it keeps cytosine methylation under check and regulates TFBS retention and gene activity.

Gain of bipolar disorder-related lncRNA AP1AR-DT in mice induces depressive and anxiety-like behaviors by reducing Negr1-mediated excitatory synaptic transmission

BackgroundBipolar disorder is a complex polygenic disorder that is characterized by recurrent episodes of depression and mania, the heterogeneity of which is likely complicated by epigenetic modifications that remain to be elucidated.MethodsWe performed transcriptomic analysis of peripheral blood RNA from monozygotic (MZ) twins discordant for bipolar disorder to identify disease-associated differentially expressed long noncoding RNAs (DE-lncRNAs), which were further validated in the PsychENCODE brain RNA-seq dataset. We then performed behavioral tests, electrophysiological assays, chromatin immunoprecipitation, and PCR to investigate the function of DE-lncRNAs in the mouse and cell models. Statistical analyses were performed using GraphPad Prism 9.0 or SPSS.ResultsWe identified a bipolar disorder-associated upregulated long non-coding RNA (lncRNA), AP1AR-DT. We observed that overexpression of AP1AR-DT in the mouse medial prefrontal cortex (mPFC) resulted in a reduction of both the total spine density and the spontaneous excitatory postsynaptic current (sEPSC) frequency of mPFC neurons as well as depressive and anxiety-like behaviors. A combination of the results of brain transcriptome analysis of AP1AR-DT overexpressing mice brains with the known genes associated with bipolar disorder revealed that NEGR1, which encodes neuronal growth regulator 1, is one of the AP1AR-DT targets and is reduced in vivo upon gain of AP1AR-DT in mice. We further demonstrated that overexpression of recombinant Negr1 in the mPFC neurons of AP1AR-DTOE mice ameliorates depressive and anxiety-like behaviors and normalizes the reduced excitatory synaptic transmission induced by the gain of AP1AR-DT. We finally identified that AP1AR-DT reduces NEGR1 expression by competing for the transcriptional activator NRF1 in the overlapping binding site of the NEGR1 promoter region.ConclusionsThe epigenetic and pathophysiological mechanism linking AP1AR-DT to the modulation of depressive and anxiety-like behaviors and excitatory synaptic function provides etiological implications for bipolar disorder.

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

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.