Rosa canina extract relieves methylation alterations of pancreatic genes in STZ-induced diabetic rats : Gene methylation in diabetic rats treated with an extract.

BACKGROUND: Diabetes is a chronic metabolic disease that affects many parts of the body. Considering diabetes as a beta cells' defect and loss, the focus is on finding mechanisms and compounds involved in stimulating the function and regeneration of pancreatic ß-cells. DNA methylation as an epigenetic mechanism plays a pivotal role in the ß-cells' function and development. Considering the regenerative and anti-diabetic effects of Rosa canina extract, this study aimed to assess the methylation levels of Pdx-1, Pax-4, and Ins-1 genes in diabetic rats treated with Rosa Canina extract. METHODS AND RESULTS: Streptozotocin-induced diabetic rats were used to evaluate the frequency of Pdx-1, Pax-4, and Ins-1 gene methylation. Treatment groups were exposed to Rosa canina as spray-dried and decoction extracts. Following blood glucose measurement, pancreatic DNA was extracted and bisulfited. Genes' methylation was measured using MSP-PCR and qRT-PCR techniques. Oral administration of Rosa canina extracts significantly reduced blood sugar levels in diabetic rats compared to the control group. The methylation levels of the Pdx-1, Pax-4, and Ins-1 genes promoter in streptozotocin-induced diabetic rats increased compared to the control rats while, the treatment of diabetic rats with Rosa canina extracts, spray-dried samples especially, led to a decreased methylation in these genes. CONCLUSION: The results of this study showed that Rosa canina extract as a spray-dried sample could be effective in treating diabetes by regulating the methylation of genes including Pdx-1, Pax-4, and Ins-1 involved in the activity and regeneration of pancreatic islet cells.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Epigenetic Regulation of Carbonic Anhydrase 9 Expression by Nitric Oxide in Human Small Airway Epithelial Cells.

DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.

The Evaluation of the N-cadherin Promoter's ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines.

During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy. To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-ß and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and ß-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test. After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of ß-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-ß in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion. Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells.

CmERF1 acts as a positive regulator of fruits and leaves growth in melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an important horticultural and economic crop. ETHYLENE RESPONSE FACTOR1 (ERF1) plays an important role in regulating plant development, and the resistance to multiple biotic and abiotic stresses. In this study, developmental biology, molecular biology and biochemical assays were performed to explore the biological function of CmERF1 in melon. Abundant transcripts of CmERF1 were found in ovary at green-yellow bud (GYB) and rapid enlargement (ORE) stages. In CmERF1 promoter, the cis-regulatory elements for indoleacetic acid (IAA), methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), gibberellic acid (GA), light and low temperature responses were found. CmERF1 could be significantly induced by ethylene, IAA, MeJA, SA, ABA, and respond to continuous light and low temperature stresses in melon. Ectopic expression of CmERF1 increased the length of siliqua and carpopodium, and expanded the size of leaves in Arabidopsis. Knockdown of CmERF1 led to smaller ovary at anthesis, mature fruit and leaves in melon. In CmERF1-RNAi #2 plants, 75 genes were differently expressed compared with control, and the promoter regions of 28 differential expression genes (DEGs) contained the GCC-box (AGCCGCC) or DRE (A/GCCGAC) cis-acting elements of CmERF1. A homolog of cell division cycle protein 48 (CmCDC48) was proved to be the direct target of CmERF1 by the yeast one-hybrid assay and dual-luciferase (LUC) reporter (DLR) system. These results indicated that CmERF1 was able to promote the growth of fruits and leaves, and involved in multiple hormones and environmental signaling pathways in melon.

Vernalization promotes bolting in sugar beet by inhibiting the transcriptional repressors of BvGI.

Sugar beet (Beta vulgaris L.), a biennial sugar crop, contributes about 16% of the world's sugar production. The transition from vegetative growth, during which sugar accumulated in beet, to reproductive growth, during which sugar exhausted in beet, is determined by vernalization and photoperiod. GIGANTEA (GI) is a key photoperiodic flowering gene that is induced by vernalization in sugar beet. To identify the upstream regulatory factors of BvGI, candidate transcription factors (TF) that were co-expressed with BvGI and could bind to the BvGI promoter were screened based on weighted gene co-expression network analysis (WGCNA) and TF binding site prediction. Subsequently, their transcriptional regulatory role on the BvGI was validated through subcellular localization, dual-luciferase assays and yeast transformation tests. A total of 7,586 differentially expressed genes were identified after vernalization and divided into 18 co-expression modules by WGCNA, of which one (MEcyan) and two (MEdarkorange2 and MEmidnightblue) modules were positively and negatively correlated with the expression of BvGI, respectively. TF binding site predictions using PlantTFDB enabled the screening of BvLHY, BvTCP4 and BvCRF4 as candidate TFs that negatively regulated the expression of BvGI by affecting its transcription. Subcellular localization showed that BvLHY, BvTCP4 and BvCRF4 were localized to the nucleus. The results of dual-luciferase assays and yeast transformation tests showed that the relative luciferase activity and expression of HIS3 was reduced in the BvLHY, BvTCP4 and BvCRF4 transformants, which suggested that the three TFs inhibited the BvGI promoter. In addition, real-time quantitative reverse transcription PCR showed that BvLHY and BvTCP4 exhibited rhythmic expression characteristics similar to that of BvGI, while BvCRF4 did not. Our results revealed that vernalization crosstalked with the photoperiod pathway to initiate bolting in sugar beet by inhibiting the transcriptional repressors of BvGI.

Callus-specific CRISPR/Cas9 system to increase heritable gene mutations in maize.

MAIN CONCLUSION: A callus-specific CRISPR/Cas9 (CSC) system with Cas9 gene driven by the promoters of ZmCTA1 and ZmPLTP reduces somatic mutations and improves the production of heritable mutations in maize. The CRISPR/Cas9 system, due to its editing accuracy, provides an excellent tool for crop genetic breeding. Nevertheless, the traditional design utilizing CRISPR/Cas9 with ubiquitous expression leads to an abundance of somatic mutations, thereby complicating the detection of heritable mutations. We constructed a callus-specific CRISPR/Cas9 (CSC) system using callus-specific promoters of maize Chitinase A1 and Phospholipid transferase protein (pZmCTA1 and pZmPLTP) to drive Cas9 expression, and the target gene chosen for this study was the bZIP transcription factor Opaque2 (O2). The CRISPR/Cas9 system driven by the maize Ubiquitin promoter (pZmUbi) was employed as a comparative control. Editing efficiency analysis based on high-throughput tracking of mutations (Hi-TOM) showed that the CSC systems generated more target gene mutations than the ubiquitously expressed CRISPR/Cas9 (UC) system in calli. Transgenic plants were generated for the CSC and UC systems. We found that the CSC systems generated fewer target gene mutations than the UC system in the T0 seedlings but reduced the influence of somatic mutations. Nearly 100% of mutations in the T1 generation generated by the CSC systems were derived from the T0 plants. Only 6.3-16.7% of T1 mutations generated by the UC system were from the T0 generation. Our results demonstrated that the CSC system consistently produced more stable, heritable mutants in the subsequent generation, suggesting its potential application across various crops to facilitate the genetic breeding of desired mutations.

The transcriptional regulation of a putative hemicellulose gene, PtrPARVUS2 in poplar.

The plant cell wall serves as a critical interface between the plant and its environment, offering protection against various stresses and contributing to biomass production. Hemicellulose is one of the major components of the cell wall, and understanding the transcriptional regulation of its production is essential to fully understanding cell wall formation. This study explores the regulatory mechanisms underlying one of the genes involved in hemicellulose biosynthesis, PtrPARVUS2. Six transcription factors (TFs) were identified from a xylem-biased library to negatively regulate PtrPARVUS2 expression. These TFs, belonging to diverse TF families, were confirmed to bind to specific cis-elements in the PtrPARVUS2 promoter region, as validated by Yeast One-Hybrid (Y1H) assays, transient expression analysis, and Chromatin Immunoprecipitation sequencing (ChIP-seq) assays. Furthermore, motif analysis identified putative cis-regulatory elements bound by these TFs, shedding light on the transcriptional regulation of SCW biosynthesis genes. Notably, several TFs targeted genes encoding uridine diphosphate glycosyltransferases (UGTs), crucial enzymes involved in hemicellulose glycosylation. Phylogenetic analysis of UGTs regulated by these TFs highlighted their diverse roles in modulating hemicellulose synthesis. Overall, this study identifies a set of TFs that regulate PARVUS2 in poplar, providing insights into the intricate coordination of TFs and PtrPARVUS2 in SCW formation. Understanding these regulatory mechanisms enhances our ability to engineer plant biomass for tailored applications, including biofuel production and bioproduct development.

Structural basis of promoter recognition by Staphylococcus aureus RNA polymerase.

Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σA controls housekeeping functions, S. aureus σB regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the -35 element and -10 element of σB regulated promoters are shorter than those of σA regulated promoters. Therefore, how σB recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σA and σB, respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the -10 element of σA regulated promoters is recognized by domain σA2 as single-stranded DNA, the -10 element of σB regulated promoters is co-recognized by domains σB2 and σB3 as double-stranded DNA, accounting for the short spacers of σB regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP.

Genome-wide identification and expression analysis of the KNOX family and its diverse roles in response to growth and abiotic tolerance in sweet potato and its two diploid relatives.

KNOXs, a type of homeobox genes that encode atypical homeobox proteins, play an essential role in the regulation of growth and development, hormonal response, and abiotic stress in plants. However, the KNOX gene family has not been explored in sweet potato. In this study, through sequence alignment, genomic structure analysis, and phylogenetic characterization, 17, 12 and 11 KNOXs in sweet potato (I. batatas, 2n = 6x = 90) and its two diploid relatives I. trifida (2n = 2x = 30) and I. triloba (2n = 2x = 30) were identified. The protein physicochemical properties, chromosome localization, phylogenetic relationships, gene structure, protein interaction network, cis-elements of promoters, tissue-specific expression and expression patterns under hormone treatment and abiotic stresses of these 40 KNOX genes were systematically studied. IbKNOX4, -5, and - 6 were highly expressed in the leaves of the high-yield varieties Longshu9 and Xushu18. IbKNOX3 and IbKNOX8 in Class I were upregulated in initial storage roots compared to fibrous roots. IbKNOXs in Class M were specifically expressed in the stem tip and hardly expressed in other tissues. Moreover, IbKNOX2 and - 6, and their homologous genes were induced by PEG/mannitol and NaCl treatments. The results showed that KNOXs were involved in regulating growth and development, hormone crosstalk and abiotic stress responses between sweet potato and its two diploid relatives. This study provides a comparison of these KNOX genes in sweet potato and its two diploid relatives and a theoretical basis for functional studies.

Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers.

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the "simultaneous inhibition of a paralog pair" concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system.

Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory.

Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Analysis of PD-L1 promoter methylation combined with immunogenic context in pancreatic ductal adenocarcinoma.

Despite the successful application of programmed cell death ligand 1 (PD-L1)-blocking strategies in some types of cancers and well-established prognostic indicators in pancreatic ductal adenocarcinoma (PDAC), the biological and clinical implications of the methylation status of PD-L1/PD-L2 in PDAC remain largely unknown. Therefore, this study aimed to explore the biological role of PD-L1/PD-L2 methylation and its association with clinicopathological features, clinical outcomes, and the immune microenvironment by analyzing the data on PD-L1/PD-L2 methylation and mRNA expression in PDAC cohorts obtained from the Cancer Genome Atlas and International Cancer Genome Consortium. The correlation between PD-L1 promoter methylation and PD-L1 expression and survival was further validated in an independent validation cohort (Peking Union Medical College Hospital [PUMCH] cohort) using pyrosequencing and immunohistochemistry. These results demonstrated that hypomethylation of the PD-L1 promoter was strongly associated with upregulated PD-L1 expression and shorter overall survival in PDAC. Multivariate Cox regression analyses revealed that the PD-L1 promoter methylation was an independent prognostic factor. PD-L1 promoter hypomethylation and high expression were related to aggressive clinical phenotypes. Moreover, both PD-L1 and PD-L2 methylation correlated with immune cell infiltration and the expression of immune checkpoint genes. PD-L1 promoter methylation status was further validated as an independent prognostic biomarker in patients with PDAC using the PUMCH cohort. The prognostic significance of PD-L1 promoter methylation was more discriminative in tumors with perineural/lymphovascular invasion and distant metastasis than in those without perineural/lymphovascular invasion and distant metastasis. In summary, the methylation status of the PD-L1 promoter is a promising biomarker for survival outcomes, immune infiltration, and the potential immune benefits of immunotherapy in PDAC.

Genome-wide identification of the MED25 BINDING RING-H2 PROTEIN gene family in foxtail millet (Setaria italica L.) and the role of SiMBR2 in resistance to abiotic stress in Arabidopsis.

MAIN CONCLUSION: The SiMBR genes in foxtail millet were identified and studied. Heterologous expression of SiMBR2 in Arabidopsis can improve plant tolerance to drought stress by decreasing the level of reactive oxygen species. Foxtail millet (Setaria italica L.), a C4 crop recognized for its exceptional resistance to drought stress, presents an opportunity to improve the genetic resilience of other crops by examining its unique stress response genes and understanding the underlying molecular mechanisms of drought tolerance. In our previous study, we identified several genes linked to drought stress by transcriptome analysis, including SiMBR2 (Seita.7G226600), a member of the MED25 BINDING RING-H2 PROTEIN (MBR) gene family, which is related to protein ubiquitination. Here, we have identified ten SiMBR genes in foxtail millet and conducted analyses of their structural characteristics, chromosomal locations, cis-acting regulatory elements within their promoters, and predicted transcription patterns specific to various tissues or developmental stages using bioinformatic approaches. Further investigation of the stress response of SiMBR2 revealed that its transcription is induced by treatments with salicylic acid and gibberellic acid, as well as by salt and osmotic stresses, while exposure to high or low temperatures led to a decrease in its transcription levels. Heterologous expression of SiMBR2 in Arabidopsis thaliana enhanced the plant's tolerance to water deficit by reducing the accumulation of reactive oxygen species under drought stress. In summary, this study provides support for exploring the molecular mechanisms associated with drought resistance of SiMBR genes in foxtail millet and contributing to genetic improvement and molecular breeding in other crops.

Exportin 4 DNA promoter methylation in liver fibrosis.

A role for exportin 4 (XPO4) in the pathogenesis of liver fibrosis was recently identified. We sought to determine changes in hepatic XPO4 promoter methylation levels during liver fibrosis. The quantitative real-time RT-PCR technique was used to quantify the mRNA level of XPO4. Additionally, pyrosequencing was utilized to assess the promoter methylation status of XPO4. The methylation rate of the XPO4 promoter was significantly increased with fibrosis in human and mouse models, while XPO4 mRNA expression negatively correlated with methylation of its promoter. DNA methyltransferases (DNMTs) levels (enzymes that drive DNA methylation) were upregulated in patients with liver fibrosis compared to healthy controls and in hepatic stellate cells upon transforming growth factor beta (TGFß) stimulation. The DNA methylation inhibitor 5-Aza or specific siRNAs for these DNMTs led to restoration of XPO4 expression. The process of DNA methylation plays a crucial role in the repression of XPO4 transcription in the context of liver fibrosis development.

Structural and functional insights into transcription activation of the essential LysR-type transcriptional regulators.

The enormous LysR-type transcriptional regulators (LTTRs), which are diversely distributed amongst prokaryotes, play crucial roles in transcription regulation of genes involved in basic metabolic pathways, virulence and stress resistance. However, the precise transcription activation mechanism of these genes by LTTRs remains to be explored. Here, we determine the cryo-EM structure of a LTTR-dependent transcription activation complex comprising of Escherichia coli RNA polymerase (RNAP), an essential LTTR protein GcvA and its cognate promoter DNA. Structural analysis shows two N-terminal DNA binding domains of GcvA (GcvA_DBD) dimerize and engage the GcvA activation binding sites, presenting the -35 element for specific recognition with the conserved σ70R4. In particular, the versatile C-terminal domain of α subunit of RNAP directly interconnects with GcvA_DBD, σ70R4 and promoter DNA, providing more interfaces for stabilizing the complex. Moreover, molecular docking supports glycine as one potential inducer of GcvA, and single molecule photobleaching experiments kinetically visualize the occurrence of tetrameric GcvA-engaged transcription activation complex as suggested for the other LTTR homologs. Thus, a general model for tetrameric LTTR-dependent transcription activation is proposed. These findings will provide new structural and functional insights into transcription activation of the essential LTTRs.

Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.