Reprogramming Of Gene Expression Research Articles

Clocking out and letting go to unleash green biotech applications in a photosynthetic host

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Abstract PO3-24-01: Mapping inter- and intra-tumor heterogeneity in Ductal Carcinoma in situ and invasive breast cancer using integrative multi-omic profiling

Abstract Background: Molecular profiling of ductal carcinoma in situ (DCIS) has shown some prognostic utility in the clinic. However, there is still an incomplete understanding of the diversity of molecular mechanisms by which DCIS progresses to invasive breast cancer (IBC).Here, we utilize integrative multi-omic profiling of co-occurring DCIS and IBC in humans as a model for mapping the relationship between tumor mutations, global gene expression and morphological changes in DCIS and IBC. Methods: We performed targeted panel DNA sequencing (Tempus xT), whole transcriptome profiling and hyperplex immunofluorescence (Lunaphore COMET) on a cohort of 39 pairs of co-occurring DCIS and IBC lesions. We performed an integrative analysis of the above multi-modal data to uncover similar and dissimilar molecular trajectories in DCIS/IBC pairs. DAVID, KEGG and Reactome databases were utilized to analyze differential pathway activity between DCIS and IBC lesions. Results: IBC samples significantly overexpressed pathways associated with cycling cells and proliferation. For DCIS, many of the enriched pathways were related to CYP enzymes and xenobiotic metabolism. We also assessed enrichment of specific transcription factor binding sites (TFBS) with the associated differentially expressed genes. The top site enriched in IBC was a promoter regulatory element of unknown function (M120 motif KRCTCNNNNMANAGC) as well as sites regulated by heat shock transcription factor 4 (HSF4). Top TFBS enriched in DCIS included MEF2, HMEF2, AR and CEBPE. The top pathways associated with IBC using DAVID were related to extracellular matrix (ECM) organization and regulation. Interestingly, the top pathways associated with DCIS also included several pathways associated with ECM organization. On Reactome pathways diagrams for ECM formation distinct components of ECM formation and organization were enriched in IBC vs DCIS lesions, with invadopodia formation components and proteoglycans specifically enriched in IBC and integrin/laminin signaling enriched in DCIS. Among the 39 cases profiled, we observed distinct patterns of DCIS/IBC pairs with highly similar mutational profiles indicative of derivation from a shared progenitor as well as pairs with little-to-no molecular concordance that likely arose independently. Even within mutationally similar DCIS/IBC pairs there were different degrees of gene expression concordance. We subdivided mutationally similar pairs into two groups based on gene expression similarity (Euclidean Distance) and identified 115 genes that differed between these groups (q < 0.05), with the dissimilar DCIS lesions hallmarked by upregulation of a variety of pathways related to B-cell function, complement cascade and cell cycle progression. Specific mutations also were related to conserved transcriptional programs in these lesions, with MCL-1 amplified tumors showing a common gene expression signature of 357 altered genes (q < 0.05) with key differences in extracellular matrix organization and adaptive immune response pathways. Dissimilarly, tumors with CKS1B copy number alterations associated with 627 differentially expressed genes (q < 0.05) with the top targets related to endothelial function and angiogenesis. Conclusions: Integrative multi-modal analysis helps us better understand tumor progression at multiple levels of biomolecular dysregulation and shows the complex interplay among genome alterations, gene expression reprogramming and tissue morphology. Such approaches may yield future biomarker signatures that better encapsulate the diverse mechanisms by which DCIS lesions evolve. Citation Format: Henry Kaplan, Alexa Dowdell, Racheli Ben Shimoli, Angela Crabtree, Ann Berry, Fred Robinson, Christopher Carney, Carlo Bifulco, Brian Piening. Mapping inter- and intra-tumor heterogeneity in Ductal Carcinoma in situ and invasive breast cancer using integrative multi-omic profiling [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-01.

Abstract PO1-06-13: Plasma Exosomes in Obesity-driven Diabetes Exacerbate Progression of Triple Negative Breast Cancer: Insights from Animal Models

Abstract Objective: Our objective is to examine how obesity and type 2 diabetes (T2D) drive systemic and local metabolic changes in adipose tissue to promote progression of triple negative breast cancer (TNBC). We investigate the role of plasma exosomes as significant mediators of gene expression and functional changes, increasing TNBC progression and metastasis. We hypothesize that T2D and obesity alter the payload of plasma exosomes. These modified exosomes, specifically differentially expressed microRNAs in T2D, critically reshape the tumor microenvironment, ultimately promoting epithelial to mesenchymal transition (EMT) and distant metastasis of TNBC cells. Our goal is to gain a deeper understanding of the underlying mechanisms connecting T2D, obesity and TNBC metastasis, through the novel factor of exosome crosstalk. Our findings shed light on novel therapeutic targets and strategies to mitigate the impact of metabolic disorders on cancer outcomes. Methods: We subjected C57BL/6J female mice to high-fat diet (HFD; 60% Kcal fat) for 12 weeks with weight monitoring, whereupon oral glucose tolerance test confirmed glucose intolerance and insulin resistance. Plasma exosomes were then isolated from peripheral blood and used to treat E0771 cells, as a TNBC model, for 72 hours for in vitro and in vivo analysis. To assess gene expression changes, we employed a commercial EMT array kit and performed qPCR. Gene expression data were uploaded to Qiagen software, specifically the Ingenuity Pathway Analysis tool, for analysis. To evaluate cell migration as a functional test of aggressiveness, a migration assay was conducted using 24-hour FBS deprivation, followed by 24-hour migration, utilizing transwell plates with 8μm pores. For in vivo analysis, mice were injected with 50k E0771-GFP cells in the 4th mammary fat pad. Tumors were allowed to grow for 28 days, at which time histology and flow cytometry analyses were performed with harvested brain, lung, and spleen. Results: In-vitro analysis showed that HFD-derived exosomes reprogram gene expression in E0771 cells, driving a strong EMT signature, compared to LFD control or exosome-free negative control. This reprogramming elicited a pro-metastatic phenotype and upregulated signaling pathways associated with metastasis. Furthermore, HFD exosomes upregulated PD-L1 receptor expression compared to negative controls, linking EMT to immune tolerance. In-vivo analysis confirmed these findings, demonstrating increased metastatic burden in both lung and brain in the HFD context. Conclusions: Plasma exosomes derived from HFD fed mice dramatically reprogram EMT gene networks in E0771 cells, exerting genomic alterations that persist for at least 28 days. These induced changes in cellular gene expression likely promote the development of brain metastases and immune evasion. The model suggests that TNBC patients with obesity-driven diabetes should be evaluated for increased risk of distant metastases. Plasma exosomes miRNA profiling may be a valuable biomarker in cancer disparities populations where the prevalence of obesity and diabetes are high. Citation Format: Pablo Llevenes. Plasma Exosomes in Obesity-driven Diabetes Exacerbate Progression of Triple Negative Breast Cancer: Insights from Animal Models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-13.

Redox regulation of epigenetic and epitranscriptomic gene regulatory pathways in plants.

Developmental and environmental constraints influence genome expression through complex panels of regulatory mechanisms. Epigenetic modifications and remodelling of chromatin are some of the major actors regulating the dynamic of gene expression. Unravelling the factors relaying environmental signals to gene expression reprogramming under stress conditions is an important and fundamental question. Indeed, many enzymes involved in epigenetic and chromatin modifications, are regulated by redox pathways, through post-translational modifications of proteins or by modifications of the flux of metabolic intermediates. Such modifications are potential hubs to relay developmental and environmental changes for gene expression reprogramming. In this review, we aim to update the current knowledge on the interaction between major redox mediators such as ROS, RNS and antioxidant, and epigenetic changes in plants. We will detail how redox status alters the post-translational modifications of proteins, intracellular epigenetic and epitranscriptional modifications, and how redox regulation interplays with DNA methylation, histone acetylation and methylation, miRNA biogenesis, and chromatin structure and remodelling, to reprogram genome expression under environmental constraints.

Genetic and Epigenetic Mechanisms Regulating Blood Pressure and Kidney Dysfunction.

The pioneering work of Dr Lewis K. Dahl established a relationship between kidney, salt, and high blood pressure (BP), which led to the major genetic-based experimental model of hypertension. BP, a heritable quantitative trait affected by numerous biological and environmental stimuli, is a major cause of morbidity and mortality worldwide and is considered to be a primary modifiable factor in renal, cardiovascular, and cerebrovascular diseases. Genome-wide association studies have identified monogenic and polygenic variants affecting BP in humans. Single nucleotide polymorphisms identified in genome-wide association studies have quantified the heritability of BP and the effect of genetics on hypertensive phenotype. Changes in the transcriptional program of genes may represent consequential determinants of BP, so understanding the mechanisms of the disease process has become a priority in the field. At the molecular level, the onset of hypertension is associated with reprogramming of gene expression influenced by epigenomics. This review highlights the specific genetic variants, mutations, and epigenetic factors associated with high BP and how these mechanisms affect the regulation of hypertension and kidney dysfunction.

Abstract 388: Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen

Abstract The integrated stress response (ISR) is an intracellular signaling pathway that fosters cell adaptation to diverse stresses. The ISR is mediated by a family of four eIF2 kinases that each sense distinct cellular stresses. Activation of the eIF2 kinases results in reprogrammed gene expression through translational and transcriptional mechanisms that help prevent stress damage. Previously, we reported that prostate cancer (PCa) cells rely on GCN2 eIF2 kinase for maintenance of amino acid (AA) homeostasis to sustain growth (Cordova et al., 2022 eLife 11:e81083). GCN2 inhibition through pharmacological or genetic means results in decreased expression of AA transporters, depletion of intracellular AAs, and reduced growth of PCa cells in culture and mouse xenograft models. Although loss of GCN2 results in a significant reduction in PCa cell growth, minimal cell death is observed. We hypothesize that loss of GCN2 in PCa cells results in vulnerabilities that can be exploited and targeted to induce cell death. To optimize GCN2-targeted therapies for PCa, we utilized a targeted CRISPR-interference (CRISPRi) screen in castration-resistant 22Rv1 cells with functional wild type (WT) GCN2 or those with it deleted (GCN2 KO). The CRISPRi screen was performed in cells grown in culture or grown as tumor xenografts in mice and featured a guide library targeting genes that encode druggable targets, including kinases, phosphatases, and metabolic enzymes. While reduced expression of 152 genes was synthetically lethal with loss of GCN2 in the in vitro screen, more genes (202) were synthetically lethal with loss of GCN2 in vivo. Of note, reduced expression of many metabolic enzymes was synthetically lethal with GCN2 ablation only in the in vivo screen, potentially due to the abundance of nutrients in standard culture media. A group of six metabolism-related genes, identified as hits in the in vivo screen and targets of FDA-approved drugs, were selected for follow-up studies. One of these metabolic enzymes was GART, a trifunctional enzyme that carries out 3 of the 10 enzymatic steps in de novo purine biosynthesis. Based on our findings showing that GCN2 maintains AA levels to support nucleotide pools in PCa cells, we initially focused on this critical enzyme. Inhibition of GART using antifolates, including the FDA-approved Pemetrexed, resulted in minimal cell death of parental and GCN2 KO PCa cells cultured in standard culture media. However, when cultured in reduced nutrient media that better mimicked reduced nutrient availability in vivo, GART inhibition resulted in selective death of GCN2 KO cells. Our CRISPRi screening strategy uncovers new vulnerabilities in GCN2-inhibited PCa cells and reveals a general strategy to identify actionable targets with the potential to combine with GCN2 inhibitors for the treatment of PCa. Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Ronald C. Wek, Kirk A. Staschke. Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen [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 388.

Abstract 1257: CHD5 suppresses glioblastoma by inhibiting MYC

Abstract Fundamentally, cancer is a disease caused by genetic and epigenetic alterations that converge to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, median survival being approximately one year and the 5-year survival rate being only 5%. Despite decades of effort, this devastating picture has not appreciably improved and effective therapies have been elusive for GBM patients. A better understanding of GBM biology is essential for developing more effective therapies. Although several genetic alterations have been implicated in gliomagenesis, driving genetic factors responsible for GBM are still obscure, and the interplay between genetic alterations and epigenetic dysregulation is largely undocumented during the gliomagenic process. Here, we show that CHD5 — a gene mapping to the 1p36 chromosomal region that is notoriously deleted in many cancers, is recurrently deleted in over 20% of GBM cases and is further downregulated through epigenetic means. To delineate how CHD5 loss promotes gliomagenesis, we established a glioma-prone mouse model harboring conditional alleles of Pten, Trp53, and Chd5. We developed an engineered mouse neural stem cell (NSCs)-based strategy to elicit glioma formation, as NSCs are the major cell-of-origin for GBM. We show that Chd5 loss significantly impairs NSCs differentiation while promoting proliferation, transformation, and gliomagenesis in vivo. Mechanistically, Chd5 forms a Chd3-containing but Chd4-independent NuRD complex that directly binds the Myc promoter and super enhancers to govern Myc expression in normal NSCs, with the Snf2 domain of Chd5 playing the most critical chromatin regulating role in the context of GBM. Chd5 loss triggers the remodeling of chromatin, enhances accessibility, and augments Myc expression, leading to the formation of highly aggressive glioma. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. This work demonstrates the tumor suppressive role of CHD5 in gliomagenesis, providing therapeutic insights for cancers with 1p36 deletions. In parallel, this work presents a powerful strategy for rapidly interrogating gene function during gliomagenesis using engineering NSCs, and the generation of novel mouse models with classic clinical features of GBM, offering new approaches for elucidating GBM biology that can inform on therapeutic opportunities for treating patients with GBM. Citation Format: Xueqin Sherine Sun, Alea Mills. CHD5 suppresses glioblastoma by inhibiting MYC [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 1257.

Gene expression reprogramming of Pseudomonas alloputida in response to arginine through the transcriptional regulator ArgR.

Different bacteria change their life styles in response to specific amino acids. In Pseudomonas putida (now alloputida) KT2440, arginine acts both as an environmental and a metabolic indicator that modulates the turnover of the intracellular second messenger c-di-GMP, and expression of biofilm-related genes. The transcriptional regulator ArgR, belonging to the AraC/XylS family, is key for the physiological reprogramming in response to arginine, as it controls transport and metabolism of the amino acid. To further expand our knowledge on the roles of ArgR, a global transcriptomic analysis of KT2440 and a null argR mutant growing in the presence of arginine was carried out. Results indicate that this transcriptional regulator influences a variety of cellular functions beyond arginine metabolism and transport, thus widening its regulatory role. ArgR acts as positive or negative modulator of the expression of several metabolic routes and transport systems, respiratory chain and stress response elements, as well as biofilm-related functions. The partial overlap between the ArgR regulon and those corresponding to the global regulators RoxR and ANR is also discussed.

Abstract B014: CHD5 suppresses glioblastoma by inhibiting MYC

Abstract Fundamentally, cancer is a disease caused by genetic and epigenetic alterations that converge to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, median survival being approximately one year and the 5-year survival rate being only 5%. Despite decades of effort, this devastating picture has not appreciably improved and effective therapies have been elusive for GBM patients. A better understanding of GBM biology is essential for developing more effective therapies. Although several genetic alterations have been implicated in gliomagenesis, driving genetic factors responsible for GBM are still obscure, and the interplay between genetic alterations and epigenetic dysregulation is largely undocumented during the gliomagenic process. Here, we show that CHD5 — a gene mapping to the 1p36 chromosomal region that is notoriously deleted in many cancers, is recurrently deleted in over 20% of GBM cases and is further downregulated through epigenetic means. To delineate how CHD5 loss promotes gliomagenesis, we established a glioma-prone mouse model harboring conditional alleles of Pten, Trp53, and Chd5. We developed an engineered mouse neural stem cell (NSCs)-based strategy to elicit glioma formation, as NSCs are the major cell-of-origin for GBM. We show that Chd5 loss significantly impairs NSCs differentiation while promoting proliferation, transformation, and gliomagenesis in vivo. Mechanistically, Chd5 forms a Chd3-containing but Chd4-independent NuRD complex that directly binds the Myc promoter and super enhancers to govern Myc expression in normal NSCs, with the Snf2 domain of Chd5 playing the most critical chromatin regulating role in the context of GBM. Chd5 loss triggers the remodeling of chromatin, enhances accessibility, and augments Myc expression, leading to the formation of highly aggressive glioma. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. This work demonstrates the tumor suppressive role of CHD5 in gliomagenesis, providing therapeutic insights for cancers with 1p36 deletions. In parallel, this work presents a powerful strategy for rapidly interrogating gene function during gliomagenesis using engineering NSCs and highlights the generation of novel mouse models with classic clinical features of GBM, offering new approaches for elucidating GBM biology that can inform on therapeutic opportunities for treating patients with GBM. Citation Format: Xueqin Sherine Sun, Alea Mills. CHD5 suppresses glioblastoma by inhibiting MYC [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B014.

FSP1 is a predictive biomarker of osteosarcoma cells’ susceptibility to ferroptotic cell death and a potential therapeutic target

Human osteosarcoma (OS) is a relatively rare malignancy preferentially affecting long body bones which prognosis is often poor also due to the lack of effective therapies. Clinical management of this cancer basically relies on surgical removal of primary tumor coupled with radio/chemotherapy. Unfortunately, most osteosarcoma cells are resistant to conventional therapy, with the undergoing epithelial-mesenchymal transition (EMT) giving rise to gene expression reprogramming, thus increasing cancer cell invasiveness and metastatic potential. Alternative clinical approaches are thus urgently needed. In this context, the recently described ferroptotic cell death represents an attractive new strategy to efficiently kill cancer cells, since most chemoresistant and mesenchymal-shaped tumors display high susceptibility to pro-ferroptotic compounds. However, cancer cells have also evolved anti-ferroptotic strategies, which somehow sustain their survival upon ferroptosis induction. Indeed, here we show that osteosarcoma cell lines display heterogeneous sensitivity to ferroptosis execution, correlating with the mesenchymal phenotype, which is consistently affected by the expression of the well-known anti-ferroptotic factor ferroptosis suppressor protein 1 (FSP1). Interestingly, inhibiting the activity or expression of FSP1 restores cancer cell sensitivity to ferroptosis. Moreover, we also found that: i) AKRs might also contribute to resistance; ii) NRF2 enhances FSP1 expression upon ferroptosis induction; while iii) p53 contributes to the regulation of FSP1 basal expression in OS cells.In conclusion, FSP1 expression can potentially be used as a valuable predictive marker of OS sensitivity to ferroptosis and as a new potential therapeutic target.

BAMLET administration via drinking water inhibits intestinal tumor development and promotes long-term health

Though new targeted therapies for colorectal cancer, which progresses from local intestinal tumors to metastatic disease, are being developed, tumor specificity remains an important problem, and side effects a major concern. Here, we show that the protein-fatty acid complex BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) can act as a peroral treatment for colorectal cancer. ApcMin/+ mice, which carry mutations relevant to hereditary and sporadic human colorectal cancer, that received BAMLET in the drinking water showed long-term protection against tumor development and decreased expression of tumor growth-, migration-, metastasis- and angiogenesis-related genes. BAMLET treatment via drinking water inhibited the Wnt/β-catenin and PD-1 signaling pathways and prolonged survival without evidence of toxicity. Systemic disease in the lungs, livers, spleens, and kidneys, which accompanied tumor progression, was inhibited by BAMLET treatment. The metabolic response to BAMLET included carbohydrate and lipid metabolism, which were inhibited in tumor prone ApcMin/+ mice and weakly regulated in C57BL/6 mice, suggesting potential health benefits of peroral BAMLET administration in addition to the potent antitumor effects. Together, these findings suggest that BAMLET administration in the drinking water maintains antitumor pressure by removing emergent cancer cells and reprogramming gene expression in intestinal and extra-intestinal tissues.

COP1 controls light-dependent chromatin remodeling

Light is a crucial environmental factor that impacts various aspects of plant development. Phytochromes, as light sensors, regulate myriads of downstream genes to mediate developmental reprogramming in response to changes in environmental conditions. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is an E3 ligase for a number of substrates in light signaling, acting as a central repressor of photomorphogenesis. The interplay between phytochrome B (phyB) and COP1 forms an antagonistic regulatory module that triggers extensive gene expression reprogramming when exposed to light. Here, we uncover a role of COP1 in light-dependent chromatin remodeling through the regulation of VIL1 (VIN3-LIKE 1)/VERNALIZATION 5, a Polycomb protein. VIL1 directly interacts with phyB and regulates photomorphogenesis through the formation of repressive chromatin loops at downstream growth-promoting genes in response to light. Furthermore, we reveal that COP1 governs light-dependent formation of chromatin loop and limiting a repressive histone modification to fine-tune expressions of growth-promoting genes during photomorphogenesis through VIL1.

PRAMEL7 and CUL2 decrease NuRD stability to establish ground-state pluripotency

Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency.

Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.

To characterize the circadian features of the trigeminal ganglion in a mouse model of headache. Several headache disorders, such as migraine and cluster headache, are known to exhibit distinct circadian rhythms of attacks. The circadian basis for these rhythmic pain responses, however, remains poorly understood. We examined trigeminal ganglion exvivo and single-cell cultures from Per2::LucSV reporter mice and performed immunohistochemistry. Circadian behavior and transcriptomics were investigated using a novel combination of trigeminovascular and circadian models: a nitroglycerin mouse headache model with mechanical thresholds measured every 6 h, and trigeminal ganglion RNA sequencing measured every 4 h for 24 h. Finally, we performed pharmacogenomic analysis of gene targets for migraine, cluster headache, and trigeminal neuralgia treatments as well as trigeminal ganglion neuropeptides; this information was cross-referenced with our cycling genes from RNA sequencing data to identify potential targets for chronotherapy. The trigeminal ganglion demonstrates strong circadian rhythms in both exvivo and single-cell cultures, with core circadian proteins found in both neuronal and non-neuronal cells. Using our novel behavioral model, we showed that nitroglycerin-treated mice display circadian rhythms of pain sensitivity which were abolished in arrhythmic Per1/2 double knockout mice. Furthermore, RNA-sequencing analysis of the trigeminal ganglion revealed 466 genes that displayed circadian oscillations in the control group, including core clock genes and clock-regulated pain neurotransmitters. In the nitroglycerin group, we observed a profound circadian reprogramming of gene expression, as 331 of circadian genes in the control group lost rhythm and another 584 genes gained rhythm. Finally, pharmacogenetics analysis identified 10 genes in our trigeminal ganglion circadian transcriptome that encode target proteins of current medications used to treat migraine, cluster headache, or trigeminal neuralgia. Our study unveiled robust circadian rhythms in the trigeminal ganglion at the behavioral, transcriptomic, and pharmacogenetic levels. These results support a fundamental role of the clock in pain pathophysiology. Several headache diseases, such as migraine and cluster headache, have headaches that occur at the same time each day. We learned that the trigeminal ganglion, an important pain structure in several headache diseases, has a 24-hour cycle that might be related to this daily cycle of headaches. Our genetic analysis suggests that some medications may be more effective in treating migraine and cluster headache when taken at specific times of the day.

CAMTAs, a family of calmodulin-binding transcription factors, are versatile regulators of biotic and abiotic stress responses in plants

Plants, rooted in one place, are constantly subjected to diverse biotic and abiotic stresses that limit their growth and development, resulting in significant crop losses. In response to stresses, plants deploy several integrated signaling networks to rapidly reprogram gene expression thereby altering cellular processes to adapt and survive under unfavorable conditions. Among the key signaling mechanisms that plants use, calcium- and calcium/calmodulin-mediated signal transduction pathways have emerged as one of the ubiquitous players. The calcium-signaling networks include many calcium and calcium/calmodulin-binding transcription factors. In this review, we focus on the functions of a family of highly conserved calcium/calmodulin-binding Transcription Factors (TFs) called calmodulin-binding transcription activators (CAMTAs) in plants. This family of transcription factors was first identified in plants as calmodulin-binding proteins and discovered later in animals. Genetic studies in the model plant Arabidopsis and crop plants such as rice uncovered crucial roles for CAMTAs in modulating plant responses to both biotic and abiotic stresses. Depending on the type of stress, CAMTAs function as either positive or negative regulators for plant growth and stress responses. Arabidopsis CAMTA3 is the most studied member of CAMTA proteins. It modulates the expression of many key genes involved in different hormone signaling pathways and plays a central role in biotic (bacterial, fungal, and viral pathogens) as well as abiotic (cold, drought, salt, and wounding/mechanical) stress responses. Studies with many point, truncated, loss-of-function, and suppressor mutants of CAMTA3 revealed a complex regulation of its function. Here we summarize the advances in the study of the CAMTA family with a focus on CAMTA3. Further, we identify critical gaps in furthering our understanding of the molecular mechanisms by which these TFs function and discuss potential opportunities to engineer them for biotechnological applications to develop stress-resilient crops.

Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

Rationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors.

A Workflow for the Functional Characterization of Noncoding RNAs in Legume Symbiotic Bacteria.

Computational comparative genomics and, later, high-throughput transcriptome profiling (RNAseq) have uncovered a plethora of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated in response to different biotic and abiotic stimuli and have the ability to fine-tune posttranscriptional reprogramming of gene expression through protein-assisted antisense interactions with trans-encoded target mRNAs. However, this level of gene regulation is still understudied in most non-model bacteria. Here, we compile experimental methods to detect expression, determine 5'/3'-ends, assess transcriptional regulation, generate mutants, and validate candidate target mRNAs of trans-acting sRNAs (trans-sRNAs) identified in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The workflow, molecular tools, and methods are suited to investigate the function of newly identified base-pairing trans-sRNAs in phylogenetically related α-rhizobia.

Singlet Oxygen Detection by Chemiluminescence Probes in Living Cells.

Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.

Bioelectric Reprogramming of the Bone Marrow Niche in Acute Myeloid Leukemia

Background. A large body of evidence indicates that cancer is not comprised solely of malignant cells, but that it represents part of a complex network of transformed and non-transformed cells, including mesenchymal stromal cells (MSCs), immune, nervous, endothelial, stem cells and fibroblasts that, together with soluble mediators, make up the tumor microenvironment. More recently, molecular mechanisms underlying a novel system of developmental control have been described to define the role of bioelectric gradients. Differences in endogenous resting membrane potential (V mem) of non-neural cells have importance in cell-cell interactions and regulation or dysregulation in embryogenesis, regeneration patterns, and cell behavior, especially proliferation and differentiation. Recently, the same issues have been suggested to be related to cancer where abnormal depolarization of resting V mem is associated to neoplastic cells and the role of ion channels conductivity in cancer initiation and progression deserves great attention in tumor microenvironment remodeling and therapy response. Methods and results. We characterized MSCs derived from bone marrow (BM) of pediatric acute myeloid leukemia patients (AML-MSCs) and identified significant changes in ion channel expression profile, including the Voltage-gated L-type calcium channel CaV1.2. We investigated the role of CaV1.2 and documented a reduced expression of CaV1.2 channel in 14 primary AML-MSCs (<53% by flow cytometry, p<0.001) mediating more frequent spontaneous calcium oscillations with higher amplitude than those found in MSCs derived from healthy donors (h-MSCs) (21% and 7%, respectively, p<0.05). Measurement of V mem by patch clamp and a fluorescent potential-sensitive probe DiBAC revealed that AML-MSCs showed a more depolarized V mem (-14.7 mV) with respect to h-MSCs (-30.5 mV, p<0.01). We hypothesized that depolarization may play a role in the AML-MSCs gene expression and functional reprogramming. Hence, we exposed h-MSCs to AML blasts demonstrating that MSCs V mem depolarized (-11.8 mV, n=10), resembling the AML-MSCs V mem, with a concomitant decrease of CaV1.2 channel expression level (<44% by flow cytometry, p<0.01). We treated h-MSCs with depolarizing pharmacological agents (ouabain and potassium gluconate) finding that the pharmacological-induced V mem depolarization promotes a pro-leukemia cell phenotype like the AML-MSCs. Briefly, MSCs exhibited higher proliferation level (ouabain, 2.5-fold; Kgluc, 1.9-fold, p<0.05), higher release of IL-6 (1.7-fold average, p<0.05), lower immunomodulatory potential and reduced the CaV1.2 levels (<35% by flow cytometry, p<0.05) with respect to untreated h-MSCs. On the contrary, we treated AML-MSCs with hyperpolarizing agents (lubiprostone and ivermectin), which reverted their phenotype to that of h-MSCs, including lower proliferation level (ivermectin -1.9-fold, p<0.05), higher immunomodulatory potential, and higher CaV1.2 levels (1.8-fold by flow cytometry, p<0.05) with respect to untreated AML-MSCs, suggesting that depolarization is reversible and that by modeling MSCs V mem we can restore an healthy stroma within the BM niche. We transduced AML-MSCs with a CaV1.2 expressing lentiviral vector (namely re-AML-MSCs, n=7) inducing a stable hyperpolarized V mem and documented that their gene expression profile reverted toward the h-MSCs profile. Finally, we observed that re-AML-MSCs propagated their acquired bioelectrical state to the contiguous AML-MSCs establishing a healthy bio-electric field. Conclusions. We demonstrated an unprecedented AML niche bioelectrical code that governs the state of MSCs, demonstrating that the contact with AML blasts is the driving force inducing the h-MSCs V mem depolarization shift and the acquisition of pro-leukemia functions. In addition, we unveiled CaV1.2 channels as a key player in this process and postulated that rewriting the bioelectric code of the patient' AML-MSCs, through the restoration of voltage-dependent calcium channel CaV1.2 activity, could be a strategy to restore their healthy phenotype. These findings provide the biological rationale to evaluate the potential of novel MSCs gene or pharmacological therapies that could both disadvantage AML progression and restore a permissive BM niche.

Identification of cold stress-responsive microRNAs and mRNA in Eucalyptus grandis

MicroRNAs (miRNAs) are a class of non-coding endogenous small RNAs that play a crucial role in plant growth, development, morphogenesis, signal transduction, and stress response. miRNAs negatively regulate gene expression at post-transcriptional level, causing translational repression or/and transcript degradation. The role of miRNAs in understanding gene expression reprogramming under different stress types has been pointed out. Low temperature is an important factor limiting the growth and distribution of plants. Under cold stress, plants attempt to maintain homeostasis and acquire cold acclimation, which involves extensive gene expression and metabolism reprogramming. Particularly, cold stress limits the distribution and productivity of Eucalyptus plantations, significantly affecting the survival and yield of seedlings. Despite the consensus on the relevance of miRNAs in regulating gene expression in plants and Eucalyptus's huge importance for the forest industry worldwide, information on miRNAs in Eucalyptus is still very scarce. In this work, we studied the miRNA and mRNA response to cold stress in E. grandis, one of Uruguay's most widely planted Eucalyptus species. Since seedlings are more susceptible to chilling injury, we subjected 3 different genotypes of seedlings to a cold shock followed by a 12-hour recovery phase at 25°C, analyzing a total of 4 treatments (G1: control, G2: 12 hours at 4°C, G3: 24 hours at 4°C, and G4: 24 hours at 4°C + 12 hours at 25°C). Cold treatment was evaluated measuring the electrolyte leakage. The membrane's damage was verified at 12 hours at 4°C, increasing the parameter at 24 hours. The membrane was partially recovered after 12 hours at 25°C, decreasing the electrolyte leakage. Transcriptome analysis showed an increase in the expression of genes belonging to the AP2|ERF family, including the DREB1|CBF genes —a group of genes that control the activation of protection mechanisms under cold stress. Within down-regulated genes, GO terms related to photosynthesis were enriched. Several conserved and new microRNAs were identified, but only a minority changed their expression with cold treatment, among them, the miR828. This microRNA was down-regulated after the cold treatment, which, according to several studies, induces the expression of anthocyanins. Anthocyanin levels are known to increase under different conditions, including cold stress. These results provided data on gene silencing mechanisms in E. grandis and allowed characterizing the response to cold mediated by small RNAs. This data could be potentially used by selection and improvement programs seeking to select clones more tolerant to cold stress.