Promoter Sequences Research Articles

Tobacco Transcription Factor NtWRKY70b Facilitates Leaf Senescence via Inducing ROS Accumulation and Impairing Hydrogen Sulfide Biosynthesis.

Leaf senescence is the terminal stage of leaf development, and its initiation and progression are closely controlled by the integration of a myriad of endogenous signals and environmental stimuli. It has been documented that WRKY transcription factors (TFs) play essential roles in regulating leaf senescence, yet the molecular mechanism of WRKY-mediated leaf senescence still lacks detailed elucidation in crop plants. In this study, we cloned and identified a tobacco WRKY TF gene, designated NtWRKY70b, acting as a positive regulator of natural leaf senescence. The expression profile analysis showed that NtWRKY70b transcript levels were induced by aging and hydrogen peroxide (H2O2) and downregulated upon hydrogen sulfide (H2S) treatment. The physiological and biochemical assays revealed that overexpression of NtWRKY70b (OE) clearly promoted leaf senescence, triggering increased levels of reactive oxygen species (ROS) and decreased H2S content, while disruption of NtWRKY70b by chimeric repressor silencing technology (SRDX) significantly delayed the onset of leaf senescence, leading to a decreased accumulation of ROS and elevated concentration of H2S. The quantitative real-time PCR analysis showed that the expression levels of various senescence-associated genes and ROS biosynthesis-related genes (NtRbohD and NtRbohE) were upregulated in OE lines, while the expression of H2S biosynthesis-related genes (NtDCD and NtCYSC1) were inhibited in OE lines. Furthermore, the Yeast one-hybrid analysis (Y1H) and dual luciferase assays showed that NtWRKY70b could directly upregulate the expression of an ROS biosynthesis-related gene (NtRbohD) and a chlorophyll degradation-related gene (NtPPH) by binding to their promoter sequences. Accordingly, these results indicated that NtWYKY70b directly activated the transcript levels of NtRbohD and NtPPH and repressed the expression of NtDCD and NtCYCS1, thereby promoting ROS accumulation and impairing the endogenous H2S production, and subsequently accelerating leaf aging. These observations improve our knowledge of the regulatory mechanisms of WRKY TFs controlling leaf senescence and provide a novel method for ensuring high agricultural crop productivity via genetic manipulation of leaf senescence in crops.

BnAP2-12 overexpression delays ramie flowering: evidence from AP2/ERF gene expression.

The APETALA2/ethylene response factor (AP2/ERF) superfamily plays a significant role in regulating plant gene expression in response to growth and development. To date, there have been no studies into whether the ramie AP2/ERF genes are involved in the regulation of flower development. Here, 84 BnAP2/ERF members were identified from the ramie genome database, and various bioinformatics data on the AP2/ERF gene family, structure, replication, promoters and regulatory networks were analysed. BnAP2-12 was transferred into Arabidopsis through the flower-dipping method. Phylogenetic analysis classified the 84 BnAP2/ERF members into four subfamilies: AP2 (18), RAV (3), ERF (42), and DREB (21). The functional domain analysis of genes revealed 10 conserved motifs. Genetic mapping localised the 84 members on 14 chromosomes, among which chromosomes 1, 3, 5, and 8 had more members. Collinearity analysis revealed that 43.37% possibly resulted from replication events during the evolution of the ramie genome. Promoter sequence analysis identified classified cis-acting elements associated with plant growth and development, and responses to stress, hormones, and light. Transcriptomic comparison identified 3,635 differentially expressed genes (DEGs) between male and female flowers (1,803 and 1,832 upregulated and downregulated genes, respectively). Kyoto Encyclopaedia of Genes and Genomes pathway analysis categorised DEGs involved in metabolic pathways and biosynthesis of secondary metabolites. Gene Ontology enrichment analysis further identified enriched genes associated with pollen and female gamete formations. Of the 84 BnAP2/ERFs genes, 22 and 8 upregulated and downregulated genes, respectively, were present in female flowers. Co-expression network analysis identified AP2/ERF members associated with flower development, including BnAP2-12. Subcellular localisation analysis showed that the BnAP2-12 protein is localised in the nucleus and cell membrane. Overexpression BnAP2-12 delayed the flowering time of Arabidopsis thaliana. These findings provide insights into the mechanism of ramie flower development.

Abstract 576: The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for Phase 1a clinical trials in USA and is currently under clinical evaluation. RNA-seq whole-genome transcriptome analysis revealed that QN-302 selectively targets G-quadruplexes in cancer-related genes, notably by forming stable G-quadruplex complexes with appropriate promoter sequences. This results in the transcriptional down regulation of affected genes, which map to for example, the hedgehog, axon guidance, WNT/β-catenin and P38 signal transduction pathways. In many instances individual expression changes are modest, though statistically significant: cumulatively, they affect whole pathways. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their genes products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Cellular transcriptome data on these eleven genes is presented here, which together with earlier reports on them, supports the designation of QN-302 as a pan-G-quadruplex agent targeting a group of major genes involved in PDAC maintenance and metastasis. Data from a xenograft model for pancreatic cancer is also available for four of these genes, which concurs with the cellular data. We suggest that the identification of these 11 genes may also provide a basis for biomarker selection and possible patient stratification based on transcriptome data. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 576.

Abstract 1694: Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways

Abstract Earli is developing a highly sensitive, orthogonal approach that is designed to be delivered via IV injection and uses a genetic construct to usurp dysregulated pathways, and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. The key element of our cancer-activated construct that drives specificity and sensitivity of expression is a promoter sequence, synthetically engineered to leverage transcription factors (TFs) activated in cancer pathways. Acting as a molecular sensor for dysregulated TF levels, the DNA construct expresses a measurable biomarker in cancer cells, while remaining transcriptionally silent in normal adjacent tissues and benign lesions. Previously, we described a multiomics factor analysis (MOFA) to interrogate 200 non-small cell lung cancer (NSCLC) and matched normal adjacent tissue samples comprised of RNA-seq based transcriptional profiling, and MS-based proteomics and phospho-proteomics from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset, and generated a list of top TFs dysregulated in NSCLC. In this study, we show the capability to design, engineer, and test cancer specific activity of combinations of response elements using a Massively Parallel Reporter Assay (MPRA) high-throughput pooled screening (HTS) method. More than 2,000 unique sequences of homotypic or heterotypic TF binding sites corresponding to the top 100 TFs were arrayed with diverse spacing and individually barcoded for the high-throughput screens in NSCLC cell lines. To validate our top hits, we individually tested 10 sequences from the top 25% and 8 sequences from the bottom 25%. Nine of the top sequences produced signals 5-10X higher than the background, whereas only one of the eight bottom sequences produced signal higher than the background. We have individually validated 30 different response elements and found that 28 of 30 drove the expected expression, with the majority of response elements (n=20) showing expression 5-10X higher than background. Remarkably, two of the engineered response elements were able to drive expression 20-30X higher in a specific cell line activated by WNT-signaling, suggesting that these response elements can sense specific cancer dysregulated pathways. Our lead response elements are being tested across a broad range of in vitro primary tumor lines with diverse genetic backgrounds and transcriptional profiles prior to in vivo testing using our imaging platform to distinguish malignant lung nodules. In summary, we have established a robust screening platform to identify response elements that can be activated by cancer dysregulated pathways. We are currently expanding this platform to other cancer models and indications, and leveraging this to explore more complex response elements that use combinations of TFs and natural spacing. Citation Format: Elizabeth Stroebele, Yue (Wendy) Zhang, Ishan Podar, Chloe Xia, Ajda Rojc, Dariusz Wodziak, Maggie Louie, David Suhy. Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1694.

Abstract 7290: Novel plasmid-based screening method for cervical cancer

Abstract Introduction: Alabama (AL) is tied for third-highest incidence rate and has second-highest death rate of cervical cancer as of 2020. Globally, cervical cancer has the fourth-highest incidence among cancers in women; 90% of deaths from cervical cancer in 2020 occurred in low- and middle-income (LMIC) countries. Inhibitor of differentiation/DNA binding 1 (Id1) expression correlates with tumorigenesis, clinical grade/stage, and aggressiveness of cervical cancer. Thus, Id1 expression could be exploited in novel screening approaches that aim to improve survival rates of cervical cancer in LMIC and clinically underserved settings. As a first step in developing an Id1-based screening approach, these preclinical studies utilized a plasmid that induces expression of a secreted reporter enzyme (secreted embryonic alkaline phosphatase, SEAP), controlled by the Id1 promoter sequence (pId1-SEAP). These studies examined relationships between Id1 expression and kinetics of reporter expression following plasmid transfection to detect human cervical cancer cells. Methods and Results: To confirm the relevance of Id1 as a target in cervical cancer, immunohistochemical staining for Id1 was performed on a human cervical cancer tissue microarray. Western blotting was performed to determine Id1 expression in human cervical cancer cell lines (HeLa, SiHa, CaSki). Optimal transfection conditions with pId1-SEAP were determined experimentally for each line, and then media was sampled through a 48-hour period post-transfection to quantify reporter SEAP expression. Immunohistochemical stain for Id1 showed that early stage and late stage human cervical cancer tissues expressed significantly (p < 0.05) higher levels of Id1 compared to normal tissues, with stain optical density values being 3e5±1e5 for early and late stage compared to 3e4±3e4 for normal tissues (early stage p=0.0001, late stage p=0.0002). Western blot for Id1 in HeLa, SiHa, and CaSki lines showed that all three positively expressed Id1, with Beta-actin-normalized expression values of 0.78±0.12, 1.12±0.05, and 0.76±0.08, respectively. The cell lines transfected with pId1-SEAP expressed SEAP at rates significantly higher than control cells, with the highest rates of production seen during the 36-48-hour post-transfection period. Rates of SEAP production (counts/hour) during this time period were 1e4±3e3 (p=0.0278), 8e2±1e2 (p=0.0834), and 3e2±5e1 (p=0.0683) for transfected HeLa, SiHa, and CaSki cells, respectively. Conclusions: The pId1-SEAP system enabled sensitive detection of cervical cancer cells. These preliminary data support future studies evaluating the in vivo performance of the system in preclinical models of early stage cervical cancer. Further development of this approach could enable sensitive and reliable detection of cervical cancer during early stages when treatment is most effective. Citation Format: Abbigael Eli, Yolanda Hartman, Benjamin B. Kasten, Rebecca Arend, Jason Warram. Novel plasmid-based screening method for cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7290.

Abstract 1442: Establishing a papillary renal cell carcinoma mouse model with activating Met mutation

Abstract Renal cell carcinoma (RCC) is a heterogeneous group of cancers of the nephron. Papillary renal cell carcinoma (PRCC) is the second most common type of RCC. c-Met activating mutations have been associated with Type 1 PRCC which are made up of small cells containing basophilic cytoplasm covering papillae and tubular structures. In patients with PRCC, c-MetH1112Y missense mutations are associated with both somatic and germline mutations. Previous functional studies indicate codon H1112 plays an important structural role in maintaining c-Met in the inactive conformation and alterations at this codon, including H1112Y, likely disrupt this interaction facilitating constitutive activation with increased c-MET activity. A c-MetH1112Y (c-MetHY) transgene with tetO promoter sequence was micro-injected into the mouse oocyte. Transgenic mice carrying this transgene have the ability to express this activated Met protein in cells expressing rtTA (reverse tetracycline-controlled transactivator) and when doxycycline is present. Mutant mice harboring this transgene along with additional trp53 mutations showed glomeruloid like Type 1 PRCC. Our in vivo studies confirmed the expression of c-MetHY gene within the kidney and Lrp2 and Ki67 staining indicated that PRCC arises from PT (proximal tubule). PRCC canonical markers CK7, AMACR and MET expression confirmed the Type1 PRCC in our mouse model. Additionally Pan-CK, E-Cad positive expression in mutant mice further confirmed PRCC arises from epithelial cells. We have generated and described type 1 PRCC mouse model with activating-MetHY mutation. This mouse model will help illustrate the oncogenic mechanism of Met activation and identify the critical events during progression of PRCC. Citation Format: Preet Lal, Li Ding, Feng Chen. Establishing a papillary renal cell carcinoma mouse model with activating Met mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1442.

Abstract 5775: Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation

Abstract Overexpression of the receptor tyrosine kinase PDGFR-β promotes cancer growth and metastasis. PDGFR-β is a validated cancer target but PDGFR-β kinase inhibitors suffer from lack of selectivity and side effects. G-quadruplexes are an exciting class of non-B DNA secondary structures with functional importance. The G-quadruplex formed in the PDGFR-β promoter is a transcription repressor and targetable by small molecules. Therefore, the PDGFR-β promoter G-quadruplex is an attractive molecular target for anticancer drugs. Structural information of the PDGFR-β promoter G-quadruplex is crucial for understanding its cellular function and rational drug design. Herein, we used nuclear magnetic resonance (NMR) spectroscopy and biophysical methods to determine the structure and dynamics of the major G-quadruplex formed in the human PDGFR-β promoter. Unlike canonical G-quadruplexes formed by four runs of three continuous guanines (G), the major PDGFR-β G-quadruplex adopts a novel broken-strand structure where a two-G segment is continued by a non-adjacent guanine. The broken-strand G-quadruplex has a vacancy-G-tetrad (vG4) which is filled-in intramolecularly by a distal guanine. Interestingly, we discovered that the major PDGFR-β G-quadruplex is a novel mixture of two equilibrating broken-strand G-quadruplexes. Both structures adopt overall parallel-stranded folding with all 1-nt chain-reversal loops. However, a different 3’-filled-in guanine is delivered by a lateral loop, which forms structurally similar hairpin with a unique capping G-G base-pair. The formation of the equilibrating broken-strand G-quadruplexes and unique capping structures are both specific to the PDGFR-β promoter sequence. Intriguingly, the observed dynamic equilibrium between the two co-existing structures with different intramolecular fill-in guanines indicates a novel vG4 intermediate, which could be filled-in by cellular guanine metabolites such as cGMP. Therefore, our study provides critical insights into how the dynamics of the major PDGFR-β G-quadruplex lead to vG4 formation and potential regulation of gene transcription by cellular guanine metabolites. The determined molecular structures of the novel broken-strand G-quadruplexes provide a structural basis for rational design of small molecules to specifically target the major PDGFR-β promoter G-quadruplex for cancer therapeutics. Citation Format: Yichen Han, Jonathan Dickerhoff, Danzhou Yang. Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5775.

The RopGEF Gene Family and Their Potential Roles in Responses to Abiotic Stress in Brassica rapa.

Guanine nucleotide-exchange factors (GEFs) genes play key roles in plant root and pollen tube growth, phytohormone responses, and abiotic stress responses. RopGEF genes in Brassica rapa have not yet been explored. Here, GEF genes were found to be distributed across eight chromosomes in B. rapa and were classified into three subfamilies. Promoter sequence analysis of BrRopGEFs revealed the presence of cis-elements characteristic of BrRopGEF promoters, and these cis-elements play a role in regulating abiotic stress tolerance and stress-related hormone responses. Organ-specific expression profiling demonstrated that BrRopGEFs were ubiquitously expressed in all organs, especially the roots, suggesting that they play a role in diverse biological processes. Gene expression analysis revealed that the expression of BrRopGEF13 was significantly up-regulated under osmotic stress and salt stress. RT-qPCR analysis revealed that the expression of BrRopGEF13 was significantly down-regulated under various types of abiotic stress. Protein-protein interaction (PPI) network analysis revealed interactions between RopGEF11, the homolog of BrRopGEF9, and the VPS34 protein in Arabidopsis thaliana, as well as interactions between AtRopGEF1, the homolog of BrRopGEF13 in Arabidopsis, and the ABI1, HAB1, PP2CA, and CPK4 proteins. VPS34, ABI1, HAB1, PP2CA, and CPK4 have previously been shown to confer resistance to unfavorable environments. Overall, our findings suggest that BrRopGEF9 and BrRopGEF13 play significant roles in regulating abiotic stress tolerance. These findings will aid future studies aimed at clarifying the functional characteristics of BrRopGEFs.

Exploiting long read sequencing to detect azole fungicide resistance mutations in Pyrenophora teres using unique molecular identifiers

Resistance to fungicides is a global challenge as target proteins under selection can evolve rapidly, reducing fungicide efficacy. To manage resistance, detection technologies must be fast and flexible enough to cope with a rapidly increasing number of mutations. The most important agricultural fungicides are azoles that target the ergosterol biosynthetic enzyme sterol 14α-demethylase (CYP51). Mutations associated with azole resistance in the Cyp51 promoter and coding sequence can co-occur in the same allele at different positions and codons, increasing the complexity of resistance detection. Resistance mutations arise rapidly and cannot be detected using traditional amplification-based methods if they are not known. To capture the complexity of azole resistance in two net blotch pathogens of barley we used the Oxford Nanopore MinION to sequence the promoter and coding sequence of Cyp51A. This approach detected all currently known mutations from biologically complex samples increasing the simplicity of resistance detection as multiple alleles can be profiled in a single assay. With the mobility and decreasing cost of long read sequencing, we demonstrate this approach is broadly applicable for characterizing resistance within known agrochemical target sites.

Identification and analysis of the SAURs in Prunus sibirica

In the present study, we identified the members of the small auxin-up RNA (SAUR) gene family in Prunus sibirica and analyzed their localization, phylogeny, duplication, cis-elements in the promoter, and expression patterns. In total, 57 PsSAURs were identified, which were randomly distributed along eight chromosomes. Among them, eight and ten pairs of segmentally duplicated genes and tandem duplication genes were found in 13 and 21 PsSAURs, respectively. Phylogenetic analysis indicated that the PsSAURs were divided into five groups (Group A–E). Light-responsive, methyl jasmonate, abscisic acid, salicylic acid, and gibberellin, as well as low-temperature responsiveness, and defense and stress responsiveness were identified by analyzing PsSAURs promoter sequences. The collinearity analysis of P. sibirica SAURs and Prunus mume and Prunus persica SAURs family genes detected 35 and 59 pairs of gene pairs, respectively, and the Ks values of all collinearity gene pairs were almost less than 1. Expression pattern analysis showed that PsSAURs had different tissue and stage expression patterns. However, research on SAURs in non-model plants remains limited, and studies on the expression and function of SAURs are lacking. This study pro­vides a foundation for further investigations into the functional analyses of SAURs in P. sibirica.

Expression of Staphylococcus aureus translation elongation factor P is regulated by a stress-inducible promotor.

Translation elongation factor P, expressed by the efp gene, is a conserved protein closely related to bacterial virulence and environmental stress regulation responses, however, little is known about the efp gene expression regulations. Here, the strain of Staphylococcus aureus subsp. aureus NCTC 8325 was taken as the research object and cultured under different conditions, including different culture temperatures, pH, and antibiotics, to study the expression of the efp gene in S. aureus by qRT-PCR, the results showed that the expression of the efp gene is upregulated under high temperature (40°C), acidic (pH 5.4) or alkaline (pH 9.4) culture conditions, but upregulated early and downregulated later under the conditions of 0.5 MIC antibiotics (chloramphenicol at the final concentration of 2μg/mL and vancomycin at the final concentration of 0.25μg/mL), indicating that the efp promoter in S. aureus is inducible. The efp promoter sequence and structure in S. aureus were predicted by bioinformatics methods, and the predicted promoter was validated by constructing a promoter-probe vector and a series of promoter mutants, the results showed that the efp promoter sequence in S. aureus, named Pro, located in 1,548,179-1,548,250 of the S. aureus genome (NC_007795.1), and the sequence of - 10 element is CCTTATAGT, - 35 element is TTTACT. The results above could lay a foundation for screening transcription factors involved in the expression of the efp gene and then exploring the transcriptional regulation mechanism of EF-P in S. aureus.

KLF5 inhibits the migration and invasion in cervical cancer cell lines by regulating SNAI1.

Epithelial-mesenchymal transition (EMT) is an important biological process by which malignant tumor cells to acquire migration and invasion abilities. This study explored the role of KLF5 in the EMT process of in cervical cancer cell lines. Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that plays a key role in cell-cycle arrest and inhibition of apoptosis. However, the molecular mechanism by which KLF5 mediates the biological functions of cervical cancer cell lines has not been elucidated. Here, we focus on the potential function of ELF5 in regulating the EMT process in in vitro model of cervical cancer cell lines. Western-blot and real-time quantitative PCR were used to detect the expression of EMT-related genes in HeLa cells. MTT assays, cell scratch and Transwell assays were used to assess HeLa cells proliferation and invasion capability. Using the bioinformatics tool JASPAR, we identified a high-scoring KLF5-like binding sequence in the SNAI1 gene promoter. Luciferase reporter assays was used to detect transcriptional activity for different SNAI1 promoter truncates. After overexpressing the KLF5 gene in HeLa cells, KLF5 not only significantly inhibited the invasion and migration of HeLa cells, but also increased the expression of E-cadherin and decreased the expression of N-cadherin and MMP9. In addition, the mRNA expression of upstream regulators of E-cadherin, such as SNAI1, SLUG, ZEB1/2 and TWIST1 was also decreased. Furthermore, KLF5 inhibiting the expression of the SNAI1 gene via binding its promoter region, and the EMT of Hela cells was promoted after overexpression of the SNAI1 gene. These results indicate that KLF5 can downregulate the EMT process of HeLa cells by decreasing the expression of the SNAI1 gene, thereby inhibiting the migration and invasion of HeLa cervical cancer cells.

Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's ttest was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Identification of epigenetically regulated genes involved in plant-virus interaction and their role in virus-triggered induced resistance

BackgroundPlant responses to a wide range of stresses are known to be regulated by epigenetic mechanisms. Pathogen-related investigations, particularly against RNA viruses, are however scarce. It has been demonstrated that Arabidopsis thaliana plants defective in some members of the RNA-directed DNA methylation (RdDM) or histone modification pathways presented differential susceptibility to the turnip mosaic virus. In order to identify genes directly targeted by the RdDM-related RNA Polymerase V (POLV) complex and the histone demethylase protein JUMONJI14 (JMJ14) during infection, the transcriptomes of infected mutant and control plants were obtained and integrated with available chromatin occupancy data for various epigenetic proteins and marks.ResultsA comprehensive list of virus-responsive gene candidates to be regulated by the two proteins was obtained. Twelve genes were selected for further characterization, confirming their dynamic regulation during the course of infection. Several epigenetic marks on their promoter sequences were found using in silico data, raising confidence that the identified genes are actually regulated by epigenetic mechanisms. The altered expression of six of these genes in mutants of the methyltransferase gene CURLY LEAF and the histone deacetylase gene HISTONE DEACETYLASE 19 suggests that some virus-responsive genes may be regulated by multiple coordinated epigenetic complexes. A temporally separated multiple plant virus infection experiment in which plants were transiently infected with one virus and then infected by a second one was designed to investigate the possible roles of the identified POLV- and JMJ14-regulated genes in wild-type (WT) plants. Plants that had previously been stimulated with viruses were found to be more resistant to subsequent virus challenge than control plants. Several POLV- and JMJ14-regulated genes were found to be regulated in virus induced resistance in WT plants, with some of them poisoned to be expressed in early infection stages.ConclusionsA set of confident candidate genes directly regulated by the POLV and JMJ14 proteins during virus infection was identified, with indications that some of them may be regulated by multiple epigenetic modules. A subset of these genes may also play a role in the tolerance of WT plants to repeated, intermittent virus infections.

Genome-wide identification and expression profiling of the ABI5 gene family in foxtail millet (Setaria italica)

BackgroundABA Insensitive 5 (ABI5) is a basic leucine zipper transcription factor that crucially influences plant growth, development, and stress response. However, there is minimal research on the ABI5 family in foxtail millet.ResultsIn this study, 16 ABI5 genes were identified in foxtail millet, and their sequence composition, gene structures, cis-acting elements, chromosome positions, and gene replication events were analyzed. To more thoroughly evaluate the developmental mechanisms of the SiABI5 family during evolution, we selected three dicotyledons (S. lycopersicum, A. thaliana, F. tataricum) and three (Z. mays, O. sativa, S. bicolor) specific representative monocotyledons associated with foxtail millet for comparative homology mapping. The results showed that foxtail millet ABI5 genes had the best homology with maize. A promoter sequence analysis showed that the SiABI5s contain numerous cis-acting elements related to hormone and stress responses, indicating that the regulation of SiABI5 expression was complex. The expression responses of 16 genes in different tissues, seed germination, and ear development were analyzed. A total of six representative genes were targeted from five subfamilies to characterize their gene expression responses to four different abiotic stresses. Overexpression of SiABI5.12 confers tolerance to osmotic stress in transgenic Arabidopsis thaliana, which demonstrated the function of SiABI5 responded to abiotic stress.ConclusionsIn summary, our research results comprehensively characterized the SiABI5 family and can provide a valuable reference for demonstrating the role of SiABI5s in regulating abiotic stress responses in foxtail millet.

IPro2L-DG: Hybrid network based on improved densenet and global attention mechanism for identifying promoter sequences

The promoter is a key DNA sequence whose primary function is to control the initiation time and the degree of expression of gene transcription. Accurate identification of promoters is essential for understanding gene expression studies. Traditional sequencing techniques for identifying promoters are costly and time-consuming. Therefore, the development of computational methods to identify promoters has become critical. Since deep learning methods show great potential in identifying promoters, this study proposes a new promoter prediction model, called iPro2L-DG. The iPro2L-DG predictor, based on an improved Densely Connected Convolutional Network (DenseNet) and a Global Attention Mechanism (GAM), is constructed to achieve the prediction of promoters. The promoter sequences are combined feature encoding using C2 encoding and nucleotide chemical property (NCP) encoding. An improved DenseNet extracts advanced feature information from the combined feature encoding. GAM evaluates the importance of advanced feature information in terms of channel and spatial dimensions, and finally uses a Full Connect Neural Network (FNN) to derive prediction probabilities. The experimental results showed that the accuracy of iPro2L-DG in the first layer (promoter identification) was 94.10% with Matthews correlation coefficient value of 0.8833. In the second layer (promoter strength prediction), the accuracy was 89.42% with Matthews correlation coefficient value of 0.7915. The iPro2L-DG predictor significantly outperforms other existing predictors in promoter identification and promoter strength prediction. Therefore, our proposed model iPro2L-DG is the most advanced promoter prediction tool. The source code of the iPro2L-DG model can be found in https://github.com/leirufeng/iPro2L-DG.

Human proinsulin production in the milk of transgenic cattle.

The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. Pseudo-lentivirus containing the bovine β-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Nutrient limitation and oxidative stress induce the promoter of acetate operon in Salmonella Typhimurium.

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the β-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in β galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.

Lamin A K97E leads to NF-κB-mediated dysfunction of inflammatory responses in dilated cardiomyopathy.

Lamins are type V intermediate filament proteins underlying the inner nuclear membrane which provide structural rigidity to the nucleus, tether the chromosomes, maintain nuclear homeostasis, and remain dynamically associated with developmentally regulated regions of the genome. A large number of mutations particularly in the LMNA gene encoding lamin A/C results in a wide array of human diseases, collectively termed as laminopathies. Dilated Cardiomyopathy (DCM) is one such laminopathic cardiovascular disease which is associated with systolic dysfunction of left or both ventricles leading to cardiac arrhythmia which ultimately culminates into myocardial infarction. In this work, we have unraveled the epigenetic landscape to address the regulation of gene expression in mouse myoblast cell line in the context of the missense mutation LMNA 289A<G (Lys97Glu) that is found in DCM-afflicted patient with severe symptoms. Significant changes in H3-specific epigenetic modifications indicated a dysregulation in transcription machinery which was investigated by RNA sequencing analysis. The major pathways involved in IL-17 signaling, cellular response to interferon-beta and gamma, cytokine production, and related pathways are found to be downregulated. Analysis of the promoter sequences of the genes in the abovementioned pathways led us to the master regulator NF-κB and its regulatory network. We report here for the first time that there is a significant downregulation of the NF-κB pathway, which has been implicated in cardio-protection elsewhere. This provides a new pathophysiological explanation that correlates an LMNA mutation and dilated cardiomyopathy.