Gene Regulation In Response Research Articles

Unravelling Differential DNA Methylation Patterns in Genotype Dependent Manner under Salinity Stress Response in Chickpea.

DNA methylation is one of the epigenetic mechanisms that govern gene regulation in response to abiotic stress in plants. Here, we analyzed the role of epigenetic variations by exploring global DNA methylation and integrating it with differential gene expression in response to salinity stress in tolerant and sensitive chickpea genotypes. Genome-wide DNA methylation profiles showed higher CG methylation in the gene body regions and higher CHH methylation in the TE body regions. The analysis of differentially methylated regions (DMRs) suggested more hyper-methylation in response to stress in the tolerant genotype compared to the sensitive genotype. We observed higher enrichment of CG DMRs in genes and CHH DMRs in transposable elements (TEs). A positive correlation of gene expression with CG gene body methylation was observed. The enrichment analysis of DMR-associated differentially expressed genes revealed they are involved in biological processes, such as lateral root development, transmembrane transporter activity, GTPase activity, and regulation of gene expression. Further, a high correlation of CG methylation with CHG and CHH methylation under salinity stress was revealed, suggesting crosstalk among the methylation contexts. Further, we observed small RNA-mediated CHH hypermethylation in TEs. Overall, the interplay between DNA methylation, small RNAs, and gene expression provides new insights into the regulatory mechanism underlying salinity stress response in chickpeas.

Common evolutionary trajectory of short life-cycle in Brassicaceae ruderal weeds

Weed species are detrimental to crop yield. An understanding of how weeds originate and adapt to field environments is needed for successful crop management and reduction of herbicide use. Although early flowering is one of the weed trait syndromes that enable ruderal weeds to overcome frequent disturbances, the underlying genetic basis is poorly understood. Here, we establish Cardamine occulta as a model to study weed ruderality. By genome assembly and QTL mapping, we identify impairment of the vernalization response regulator gene FLC and a subsequent dominant mutation in the blue-light receptor gene CRY2 as genetic drivers for the establishment of short life cycle in ruderal weeds. Population genomics study further suggests that the mutations in these two genes enable individuals to overcome human disturbances through early deposition of seeds into the soil seed bank and quickly dominate local populations, thereby facilitating their spread in East China. Notably, functionally equivalent dominant mutations in CRY2 are shared by another weed species, Rorippa palustris, suggesting a common evolutionary trajectory of early flowering in ruderal weeds in Brassicaceae.

Nutritional and tissue-specific regulation of cytochrome P450 CYP711A MAX1 homologues and strigolactone biosynthesis in wheat.

Strigolactones (SLs) are a class of phytohormones regulating branching/tillering, and their biosynthesis has been associated with nutritional signals and plant adaptation to nutrient-limiting conditions. The enzymes in the SL biosynthetic pathway downstream of carlactone are of interest as they are responsible for structural diversity in SLs, particularly cytochrome P450 CYP711A subfamily members, such as MORE AXILLARY GROWTH1 (MAX1) in Arabidopsis. We identified 13 MAX1 homologues in wheat, clustering in four clades and five homoeologous subgroups. The utilization of RNA-sequencing data revealed a distinct expression pattern of MAX1 homologues in above- and below-ground tissues, providing insights into the distinct roles of MAX1 homologues in wheat. In addition, a transcriptional analysis showed that SL biosynthetic genes were systematically regulated by nitrogen supply. Nitrogen limitation led to larger transcriptional changes in the basal nodes than phosphorus limitation, which was consistent with the observed tillering suppression, as wheat showed higher sensitivity to nitrogen. The opposite was observed in roots, with phosphorus limitation leading to stronger induction of most SL biosynthetic genes compared with nitrogen limitation. The observed tissue-specific regulation of SL biosynthetic genes in response to nutritional signals is likely to reflect the dual role of SLs as rhizosphere signals and branching inhibitors.

Transcriptional regulation of OsWRKY genes in response to individual and overlapped challenges of Magnaporthe oryzae and drought in indica genotypes of rice

In rainfed rice cultivation, severe blast (Magnaporthe oryzae) infection is frequently observed following intermittent drought. We simulated the natural rainfed rice field and explored the complex transcriptional regulations behind enhanced susceptibility of rice towards blast disease. We identified eight new OsWRKY genes in the indica subspecies of rice using the updated v5.0 PlantTFDB and GRAMENE making it altogether 109 OsWRKY genes. Distribution of the OsWRKY genes in the 12 chromosomes of indica rice was uneven. Where, chromosome 1 houses maximum number of genes but chromosome 5 has the highest gene density. Motif analysis depicted presence of a new motif i.e., an autophagy protein 16 (ATG16) associated to group IIa of the OsWRKY genes. We selected twenty biotic stress-responsive OsWRKY genes to check their transcript accumulation pattern in individual and overlapping challenges of blast and drought in three indica rice cultivars viz., blast susceptible HR-12, and blast resistant Tetep (Pi54) and C101A51 (Pi2). Transcripts of most OsWRKY genes in the resistant cultivars accumulated early (6 h) and at higher levels compared to the susceptible check. Interestingly, a significant decrease in transcript accumulation of most of the blast responsive OsWRKY genes was observed in all the three cultivars when exposed to the overlapping challenges. Additionally, a significant reduction in biosynthesis of both the defense enzymes PAL and PO in blast and drought overlapped condition in the resistant cultivars indicated partial breakdown of resistance due to intermittent drought. To conclude, the present study has contributed to understanding of the relationship between the expression pattern of 20 biotic stress responsive OsWRKY genes in indica rice grown in rainfed condition coupled with intermittent drought and blast susceptibility.

Deciphering the contribution of PerR to oxidative stress defense system in Clostridium tyrobutyricum

AbstractAs a next‐generation probiotic, Clostridium tyrobutyricum, which is considered obligate anaerobe, has been extensively considered a promising candidate for future use to promote health benefits. The industrial applications were limited by its weak tolerance to oxygen stress during fermentation. Here, we enhanced the oxidative stress tolerance of C. tyrobutyricum L319 by deleting the peroxide response regulator gene perR using an endogenous CRISPR‐Cas system. The mutant strain exhibited greatly improved growth when exposed to oxygen, which also showed a shortened lag phase compared with the control strain. Consistently, a concomitant increase in butyric acid productivity from 0.08 to 0.13 g/L/h in the presence of oxygen was observed. Decreased intracellular ROS level sand NAD+/NADH ratios were also observed in the mutant strain. Furthermore, transcriptome and binding target analysis revealed that genes encoding transcriptional regulators and proteins related to redox balance were upregulated. Changes in carbohydrate, amino acid and purine metabolisms upon oxygen exposure were also observed. Importantly, phylogenomic analysis indicated that PerR may be a universal engineering target in diverse Clostridium species. These findings will contribute to the development of robust C. tyrobutyricum strains for efficient butyric acid production in the food industry.

Genome-wide identification and expression analysis of SlRR genes in response to abiotic stress in tomato.

The cytokinin two-component signal transduction system (TCS) is involved in many biological processes, including hormone signal transduction and plant growth regulation. Although cytokinin TCS has been well characterized in Arabidopsis thaliana, its role in tomato remains elusive. In this study, we characterized the diversity and function of response regulator (RR) genes, a critical component of TCS, in tomato. In total, we identified 31 RR genes in the tomato genome. These SlRR genes were classified into three subgroups (type-A, type-B and type-C). Various stress-responsive cis-elements were present in the tomato RR gene promoters. Their expression responses under pesticide treatment were evaluated by transcriptome analysis. Their expression under heat, cold, ABA, salinity and NaHCO3 treatments was further investigated by qRT-PCR and complemented with the available transcription data under these treatments. Specifically, SlRR13 expression was significantly upregulated under salinity, drought, cold and pesticide stress and was downregulated under ABA treatment. SlRR23 expression was induced under salt treatment, while the transcription level of SlRR1 was increased under cold and decreased under salt stress. We also found that GATA transcription factors played a significant role in the regulation of SlRR genes. Based on our results, tomato SlRR genes are involved in responses to abiotic stress in tomato and could be implemented in molecular breeding approaches to increase resistance of tomato to environmental stresses.

Pseudomonas putida induces resistance to Fusarium oxysporum f. sp. radicis-lycopersici in tomato plants by activating expression of defense-related genes

Plant growth-promoting rhizobacteria (PGPR) may prevent attack from pathogenic microorganisms by eliciting induced systemic resistance (ISR). In the present work, Pseudomonas putida isolate TR21/1 showed significant biological control of tomato seedlings inoculated with Fusarium oxysporum f.sp. radicis-lycopersici (FORL). Here, the SA-responsive genes PR-1, PR-4, PR-6 and CH9 were downregulated upon induction of ISR by P. putida strain TR21/1 and induced when bacterized tomato roots were inoculated with FORL. This indicates that SAR involves the accumulation of SA-responsive genes but ISR does not. Similarly, expression of ET-regulated genes such as ACO1, ACO3, ACO4 were not induced in ISR-expressing tomato roots and P. putida treatment induced only ACO2 expression suggesting that ACO2 expression is involved in ISR-expressing tomato seedlings. In contrast, the infection of ISR expressing plants by FORL strongly induced ACO3, ACO2, and ACO1 indicating the transcriptional regulation of ACO genes in response to FORL attack which may be related to possible ethylene synthesis in response to pathogen. Here P. putida treatment increased ETR1 gene expression in roots and this induction was upregulated in presence of FORL indicating that ETR1 plays a role in the protection of plants against FORL by reducing ethylene sensitivity. Activation of SA-and ET- regulated genes in bacterized plants in the presence of FORL implies that not only SA but other signals as well, may play an important role in inducing resistance.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Small RNA profiling reveals the involvement of microRNA-mediated gene regulation in response to symbiosis in raspberry.

Dark septate endophytes (DSEs) can form reciprocal symbioses with most terrestrial plants, providing them with mineral nutrients in exchange for photosynthetic products. Although the mechanism of plant-DSEs is well understood at the transcriptional level, little is known about their post-transcriptional regulation, and microRNAs (miRNAs) for the symbiotic process of DSE infestation of raspberry have not been identified. In this study, we comprehensively identified the miRNAs of DSE-infested raspberry symbiosis using Illumina sequencing. A total of 361 known miRNAs and 95 novel miRNAs were identified in the roots. Similar to other dicotyledons, most of the identified raspberry miRNAs were 21 nt in length. Thirty-seven miRNAs were differentially expressed during colonization after inoculation with Phialocephala fortinii F5, suggesting a possible role for these miRNAs in the symbiotic process. Notably, two miRNAs (miR171h and miR396) previously reported to be responsive to symbiotic processes in alfalfa also had altered expression during raspberry symbiosis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggests that miRNAs are mainly involved in regulatory mechanisms, such as biological processes, cellular metabolic processes, biosynthesis of secondary metabolites, plant-pathogen interactions, and phytohormone signaling pathways. This study revealed the potential conservation of miRNA-mediated post-transcriptional regulation in symbiotic processes among plants and provides some novel miRNAs for understanding the regulatory mechanisms of DSE-raspberry symbiosis.

Upregulation of Nrf2 Signalling and the Inhibition of Erastin-Induced Ferroptosis by Ferulic Acid in MIN6 Cells.

Ferroptosis is a regulated cell death process characterised by the iron-dependent accumulation of oxidised polyunsaturated fatty acid-containing phospholipids. Its initiation is complicated and involves reactive oxygen species (ROS) and a loss of the activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4). These play critical roles in the development of ferroptotic cell damage by lipid peroxidation. Antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of ferroptosis. This study was designed to demonstrate the protective effect of ferulic acid (FA) against oxidative stress and erastin-mediated ferroptosis in murine MIN6 cells. Cells were treated with FA or its metabolite ferulic acid 4-O-sulfate disodium salt (FAS) and 20 μM of erastin. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, iron levels were measured by inductively coupled plasma mass spectrometry (ICP-MS), ROS levels were determined by a dihydrodichlorofluorescein (H2DCF) cell-permeant probe, and glutathione and lipid peroxidation were assayed with commercially available kits. The phenolic acids enhanced cell viability in erastin-treated MIN6 cells in a dose-dependent manner. Furthermore, MIN6 cells exposed to erastin alone showed elevated levels of iron and ROS, glutathione (GSH) depletion, and lipid peroxidation (p < 0.05) compared to cells that were protected by co-treatment with FA or FAS. The treatment of MIN6 cells with FA or FAS following exposure to erastin increased the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) protein levels. Consequently, levels of its downstream antioxidant proteins, HO-1, NQO1, GCLC, and GPX4, increased. FA and FAS greatly decreased erastin-induced ferroptosis in the presence of the Nrf2 inhibitor, ML385, through the regulation of Nrf2 response genes. In conclusion, these results show that FA and FAS protect MIN6 cells from erastin-induced ferroptosis by the Nrf2 antioxidant protective mechanism.

Abstract A017: Bidirectional signaling between mitochondria and the nucleus dictates epigenetic rewiring to drive tumorigenesis

Abstract Cancer cells have acquired the ability to sense and adapt to varying conditions of nutrient sources in the tumor microenvironment. Mitochondria lay at the core of cellular metabolism, whereas the nucleus integrates cellular and environmental signals to activate gene transcription that alter cell function and cell fate. However, it remains largely unexplored how mitochondrial metabolic enzymes and nuclear transcriptional machineries communicate to drive gene regulation in response to metabolic stress. Biochemical screening identified a TCA cycle enzyme aconitase (ACO2), which functions as a critical mitochondrial regulator of de novo lipogenesis to sustain prostate tumor growth. Metabolic isotope tracing experiments revealed that the ACO2 enzyme promotes citrate biosynthesis required for de novo lipogenesis by reductive carboxylation of alpha-ketoglutarate generated from glutamine. Proteomics studies identified an acetylation mark lysine258 on ACO2 that induces increased enzymatic activity, whereas acetylation deficient Lys258Arg-ACO2 mutant was inactive, and failed to promote lipogenesis and tumor growth. To uncover nuclear signals that may stimulate ACO2 activity, we identified transcriptional suppression of a mitochondrial deacetylase SIRT3 by AR and its coregulator SRC-2, thereby preserving enhanced K258Ac mark on ACO2 facilitating increased lipogenesis. Interestingly our spontaneous bone metastatic mouse studies revealed that increased ACO2 activity regulated by the SIRT3/SRC-2 axis functions as a predominant survival factor for prostate tumors in the early stages of homing and colonization during bone metastasis, suggesting this pathway may be essential for metabolic adaptation in hostile microenvironment. Next, to investigate the biochemical control of gene transcription that may support tumor cell adaptation in the hostile microenvironment, we found a potential metabolic pathway that may support acetyl-CoA synthesis in the nucleus. Reversible enzymes IDH2 and ACO2 which can generate citrate from alpha-ketoglutarate to produce acetyl CoA were found to be localized in the nucleus. Interestingly, ablation of ACO2 and/or IDH2 was sufficient to significantly reduce several histone acetylation marks such as H3K9ac and H3K14ac. In isolated intact nucleus, these marks could be increased by addition of alpha-ketoglutarate thereby increasing the overall chromatin accessibility, however loss of ACO2 or IDH2 abrogated the effect, implying these enzymes are required to generate acetyl CoA in the nucleus independent of mitochondrial contribution. ATAC-seq analysis further revealed genetic inhibition of ACO2 significantly reduced chromatin accessibility with an approximate loss of 16626 peaks and gain of only 486 peaks compared to control (WT) cells. Our findings indicate a potential metabolic control in the nucleus that may regulate chromatin accessibility required for gene activation to favor tumor cell adaptation in a hostile microenvironment. This work is supported by the funds from NIH (R01CA252092 and DP2CA260421) to S.D. Citation Format: Abhisha Sawant Dessai, Nadya Elhalawany, Eriko Katsuta, Christian Prechtl, Spencer Rosario, Bert W. O’Malley, Xiang H. Zhang, Subhamoy Dasgupta. Bidirectional signaling between mitochondria and the nucleus dictates epigenetic rewiring to drive tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A017.

Effects of Thermal Manipulation on mRNA Regulation of Response Genes Regarding Improvement of Thermotolerance Adaptation in Chickens during Embryogenesis

The phenomenon of increasing heat stress (HS) among animals is of particular significance when it is seen in economically significant industries, such as poultry. Due to the identification of the physiological, molecular, and genetic roots of HS responses in chickens, a substantial number of studies have focused on reducing the effects of HS in poultry through environmental management, dietary manipulation, and genetic alterations. Temperature manipulation (TM) during embryogenesis has been claimed to increase the thermal tolerance and well-being of chickens without affecting their capacity for future growth. There has been little investigation into the vulnerability of the epigenome involving TM during embryogenesis, although the cellular pathways activated by HS have been explored in chickens. Epigenetic changes caused by prenatal TM enhance postnatal temperature adaption and produce physiological memory. This work offers a thorough analysis that explains the cumulative impact of HS response genes, such as genes related to heat shock proteins, antioxidants, and immunological genes, which may aid in the enhanced adaptability of chickens that have undergone thermal manipulation during their embryonic stages.

Heat shock factor HSF1 regulates BDNF gene promoters upon acute stress in the hippocampus, together with pCREB.

Heat shock factor 1 (HSF1) is a master stress-responsive transcriptional factor, protecting cells from death. However, its gene regulation in vivo in the brain in response to neuronal stimuli remains elusive. Here, we investigated its direct regulation of the brain-derived neurotrophic factor (BDNF) gene (Bdnf) in response to acute neuronal stress stimuli in the brain. The results of immunohistochemistry and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) showed that administration of kainic acid (a glutamate receptor agonist inducing excitotoxity) to young adult mice induced HSF1 nuclear translocation and its binding to multiple Bdnf promoters in the hippocampus. Footshock, a physical stressor used for learning, also induced HSF1 binding to selected Bdnf promoters I and IV. This is, to our knowledge, the first demonstration of HSF1 gene regulation in response to neuronal stimuli in the hippocampus in vivo. HSF1 binding sites (HSEs) in Bdnf promoters I and IV were also detected when immunoprecipitated by an antibody of phosphorylated (p)CREB (cAMP-responsive element-binding protein), suggesting their possible interplay in acute stress-induced Bdnf transcription. Interestingly, their promoter binding patterns differed by KA and footshock, suggesting that HSF1 and pCREB orchestrate to render fine-tuned promoter control depending on the types of stress. Further, HSF1 overexpression increased Bdnf promoter activity in a luciferase assay, while virus infection of constitutively active-form HSF1 increased levels of BDNF mRNA and protein in vitro in primary cultured neurons. These results indicated that HSF1 activation of Bdnf promoter was sufficient to induce BDNF expression. Taken together, these results suggest that HSF1 promoter-specific control of Bdnf gene regulation plays an important role in neuronal protection and plasticity in the hippocampus in response to acute stress, possibly interplaying with pCREB.

Improvement of Rice Agronomic Traits by Editing Type-B Response Regulators.

Type-B response regulator proteins in rice contain a conserved receiver domain, followed by a GARP DNA binding domain and a longer C-terminus. Some type-B response regulators such as RR21, RR22 and RR23 are involved in the development of rice leaf, root, flower and trichome. In this study, to evaluate the application potential of type-B response regulators in rice genetic improvement, thirteen type-B response regulator genes in rice were respectively knocked out by using CRISPR/Cas9 genome editing technology. Two guide RNAs (gRNAs) were simultaneously expressed on a knockout vector to mutate one gene. T0 transformed plants were used to screen the plants with deletion of large DNA fragments through PCR with specific primers. The mutants of CRISPR/Cas9 gene editing were detected by Cas9 specific primer in the T1 generation, and homozygous mutants without Cas9 were screened, whose target regions were confirmed by sequencing. Mutant materials of 12 OsRRs were obtained, except for RR24. Preliminary phenotypic observation revealed variations of various important traits in different mutant materials, including plant height, tiller number, tillering angle, heading date, panicle length and yield. The osrr30 mutant in the T2 generation was then further examined. As a result, the heading date of the osrr30 mutant was delayed by about 18 d, while the yield was increased by about 30%, and the chalkiness was significantly reduced compared with those of the wild-type under field high temperature stress. These results indicated that osrr30 has great application value in rice breeding. Our findings suggest that it is feasible to perform genetic improvement of rice by editing the type-B response regulators.

Mechanistic Study of Wee1 Kinase Inhibition with AZD1775 Exposes Drug Targetable Vulnerabilities in Acute B-Lymphoblastic Leukemia

Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side effects of chemotherapy. Despite a high response rate to induction chemotherapy in acute lymphoblastic leukemia (ALL) there is an urgent need to identify effective combination therapy to overcome treatment resistance that associates with disease relapse. ALL cells are acutely highly sensitive to the WEE1 inhibitor AZD1775 that compromises the G2/M-phase cell cycle checkpoint. Here, we show that AZD1775 selectively inhibits recovery of proliferation in KMT2A/MLL-rearranged (MLL-r) ALL, a group of leukemias that respond poorly to current chemotherapy and have dismal prognosis compared to other leukemia subgroups. This vulnerability could be distinguished by elevated WEE1 expression in MLL-r ALL and through biochemical analysis of the resulting mitotic catastrophe upon WEE1 inhibition as assessed by PARP cleavage and flow cytometric analysis of DNA metabolism. Extensive genomics profiling of the drug response by joint single-cell transcriptome and chromatin accessibility profiling revealed that AZD1775 led to a strong p53-driven gene regulatory response resulting in induction of apoptosis and senescence and disrupts a crucial hematopoietic TF-network involving RUNX1, MYC and GATA2. We further demonstrate that RUNX1 protein was degradaded in a CDK-dependent manner. Leukemia relapse may arise from rare cells that escape treatment through genetic mutations, transcriptional reprogramming or signaling rewiring. We here demonstrate that upon the pre-mature mitosis entry resulting from AZD1775 treatment, a rapid transcriptional reprogramming is triggered involving activation of NfKB and transcription factors regulating lipid metabolism, BCL6 and pre-BCR signaling that resulted in a small surviving cell population. Single-cell characterization of the AZD1775 response in a MLL-r patient-derived xenograft (PDX) model supported the in vivo relevance of this drug resistance mechanism. Informed by the genomic profiles, sequential administration of AZD1775 with inhibitors of pre-BCR signaling or fatty acid synthesis was designed to target the resistant cell state and this approach effectively prevented cell survival and recovery of ALL cells. Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell survival and cell fate regulation in response to drugs targeting cell cycle regulation and provide a rationale for combination therapies with low toxicity drugs such as dasatinib or fatostatin to counteract drug resistance that occurs through cell state switching.

Genetic and Epigenetic Cis-Regulatory Disruptions Impacting the Canonical Wnt Signaling Repressor TLE1 Gene Promote Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer and despite improved survival rates, more children die of ALL than any other cancer. Glucocorticoids (GCs) are a mainstay of contemporary, multi-drug chemotherapy in the treatment of childhood ALL. Resistance to GCs is predictive of ALL relapse and poor clinical outcomes, and therefore represents a major hurdle limiting further improvements in survival rates. While advances have been made in identifying genes implicated in GC resistance, there remains an insufficient understanding of the impact of cis-regulatory disruptions in GC resistance. This is particularly relevant for GCs, which function through the activation of the glucocorticoid receptor (GR) nuclear receptor transcription factor. To better understand the role of cis-regulatory disruptions in GC resistance we comprehensively mapped the dynamic gene regulatory response to GCs in two human ALL cell lines using functional genomic (RNA-seq, ChIP-seq, ATAC-seq and HiChIP) and massively parallel reporter (ATAC-STARR-seq) assays at multiple time points within a 24-hour window. In total, over 100 independent datasets were generated and integrated. We identified thousands of GR binding sites and GC-responsive changes to chromatin state, including the formation of over 250 GC-responsive super-enhancers and depletion of AP-1 bound cis-regulatory elements implicated in cell proliferation and anti-apoptotic processes. Our results further show that GR mediates gene regulatory effects in ALL cells at GC-responsive chromatin sites through long-range looping interactions with GC-responsive gene promoters. Notably, GC-responsive chromatin sites were also enriched for functionally active cis-regulatory elements. By integrating our GC response maps with inherited DNA sequence variants and differentially accessible chromatin sites associated with GC resistance in primary ALL cells from patients enrolled on St. Jude clinical trials, we identified two GR-bound cis-regulatory elements at the canonical Wnt signaling repressor TLE1 gene locus that were genetically or epigenetically disrupted in GC-resistant patient samples. Functional evaluation of these two GR binding sites using CRISPR genome or CRISPR interference epigenome editing validated their impact on GC resistance and TLE1 gene expression. To better understand the relationship between GC signaling and canonical Wnt signaling, we further performed TOP-flash luciferase reporter assays, cellular drug viability assays and transcriptomic drug response profiling which identified mutual antagonism between these two signaling pathways. Collectively, these data uncovered genetic and epigenetic cis-regulatory disruptions of canonical Wnt signaling as a novel mechanism impacting GC resistance in childhood ALL and further suggest that therapeutic inhibition of canonical Wnt signaling could improve GC sensitivity in patients with resistant disease.

Chromosomal and gonadal sex drive sex differences in lipids and hepatic gene expression in response to hypercholesterolemia and statin treatment

BackgroundBiological sex impacts susceptibility and presentation of cardiovascular disease, which remains the leading cause of death for both sexes. To reduce cardiovascular disease risk, statin drugs are commonly prescribed to reduce circulating cholesterol levels through inhibition of cholesterol synthesis. The effectiveness of statin therapy differs between individuals with a sex bias in the frequency of adverse effects. Limited information is available regarding the mechanisms driving sex-specific responses to hypercholesterolemia or statin treatment.MethodsFour Core Genotypes mice (XX and XY mice with ovaries and XX and XY mice with testes) on a hypercholesteremic Apoe–/– background were fed a chow diet without or with simvastatin for 8 weeks. Plasma lipid levels were quantified and hepatic differential gene expression was evaluated with RNA-sequencing to identify the independent effects of gonadal and chromosomal sex.ResultsIn a hypercholesterolemic state, gonadal sex influenced the expression levels of more than 3000 genes, and chromosomal sex impacted expression of nearly 1400 genes, which were distributed across all autosomes as well as the sex chromosomes. Gonadal sex uniquely influenced the expression of ER stress response genes, whereas chromosomal and gonadal sex influenced fatty acid metabolism gene expression in hypercholesterolemic mice. Sex-specific effects on gene regulation in response to statin treatment included a compensatory upregulation of cholesterol biosynthetic gene expression in mice with XY chromosome complement, regardless of presence of ovaries or testes.ConclusionGonadal and chromosomal sex have independent effects on the hepatic transcriptome to influence different cellular pathways in a hypercholesterolemic environment. Furthermore, chromosomal sex in particular impacted the cellular response to statin treatment. An improved understanding of how gonadal and chromosomal sex influence cellular response to disease conditions and in response to drug treatment is critical to optimize disease management for all individuals.

Role of EmaSR in Ethanol Metabolism by Acinetobacter baumannii.

Acinetobacter baumannii is a well-known nosocomial pathogen that can survive in different environments through the use of intricate networks to regulate gene expression. Two-component systems (TCS) form an important part of such regulatory networks, and in this study, we describe the identification and characterization of a novel EmaSR TCS in A. baumannii. We constructed a Tn5-tagged mutagenesis library, from which an emaS sensor kinase gene and emaR response regulator gene were identified. We found that emaS/emaR single-mutants and double-mutants were unable to replicate in M9 medium with 1% ethanol as the single carbon source. Motility and biofilm formation were negatively affected in double-mutants, and transcriptomic analysis showed that mutation of emaSR dysregulated genes required for carbon metabolism. In addition, emaS/emaR single-mutants and double-mutants were unable to survive in acetic acid- and sodium acetate-containing medium, indicating that the EmaSR TCS is also important for acetate metabolism. Furthermore, virulence against Galleria mellonella was diminished in emaS/emaR single- and double-mutants. Taken together, these results show that this novel EmaSR TCS is involved in the regulation of A. baumannii ethanol metabolism and acetate metabolism, with important implications on motility, biofilm formation, and virulence if mutated. Further research on the underlying mechanisms is warranted.

Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration.

WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture. In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.

The RAC/ROP GTPase activator OsRopGEF10 functions in crown root development by regulating cytokinin signaling in rice.

RAC/Rho of plant (ROP) GTPases are major molecular switches that control diverse signaling cascades for plant growth, development, and defense. Here, we discovered a signaling node that connects RAC/ROPs to cytokinins. Rice (Oryza sativa) plants develop a fibrous root system mainly composed of crown roots. Cytokinin signaling via a phosphorelay system is critical for crown root development. We show that OsRopGEF10, which activates RAC/ROPs, acts upstream of the cytoplasmic-nuclear shuttling phosphotransfer proteins AHPs of the cytokinin signaling pathway to promote crown root development. Mutations of OsRopGEF10 induced hypersensitivity to cytokinin, whereas overexpressing this gene reduced the cytokinin response. Loss of OsRopGEF10 function reduced the expression of the response regulator gene OsRR6, a repressor of cytokinin signaling, and impaired crown root development. Mutations in OsAHP1/2 led to increased crown root production and rescued the crown root defect of Osropgef10. Furthermore, auxin activates the ROP GTPase OsRAC3, which attenuates cytokinin signaling for crown root initiation. Molecular interactions between OsRopGEF10, OsRAC3, and OsAHP1/2 implicate a mechanism whereby OsRopGEF10-activated OsRAC3 recruits OsAHP1/2 to the cortical cytoplasm, sequestering them from their phosphorelay function in the nucleus. Together, our findings uncover the OsRopGEF10-OsRAC3-OsAHP1/2 signaling module, establish a link between RAC/ROPs and cytokinin, and reveal molecular crosstalk between auxin and cytokinin during crown root development.