Large Set Of Genes Research Articles

Enhancing Gene Expression Predictions Using Deep Learning and Functional Annotations.

Transcriptome-wide association studies (TWAS) aim to uncover genotype-phenotype relationships through a two-stage procedure: predicting gene expression from genotypes using an expression quantitative trait locus (eQTL) data set, then testing the predicted expression for trait associations. Accurate gene expression prediction in stage 1 is crucial, as it directly impacts the power to identify associations in stage 2. Currently, the first stage of such studies is primarily conducted using linear models like elastic net regression, which fail to capture the nonlinear relationships inherent in biological systems. Deep learning methods have the potential to model such nonlinear effects, but have yet to demonstrably outperform linear methods at this task. To address this gap, we propose a new deep learning architecture to predict gene expression from genotypic variation across individuals. Our method utilizes a learnable input scaling layer in conjunction with a convolutional encoder to capture nonlinear effects and higher-order interactions without compromising on interpretability. We further augment this approach to allow for parameter sharing across multiple networks, enabling us to utilize prior information for individual variants in the form of functional annotations. Evaluations on real-world genomic data show that our method consistently outperforms elastic net regression across a large set of heritable genes. Furthermore, our model statistically significantly improved predictive performance by leveraging functional annotations, whereas elastic net regression failed to show equivalent gains when using the same information, suggesting that our method can capture nonlinear functional information beyond the capability of linear models.

Abstract PR-15: Identifying synergistic combinations with KRAS inhibition in PDAC

Abstract Background: To date, targeted therapies have largely failed in eliciting sustained responses in advanced pancreatic ductal adenocarcinoma (PDAC). KRAS-targeted therapies have the potential to drastically transform clinical management. Emerging data indicate that only a subset of patients respond, and that acquired resistance is common. Here, we used a combination of in vivo CRISPR activation (CRISPRa) screening and in vitro/preclinical models to identify synergistic combinations with KRAS inhibition (KRASi) in pancreatic and lung cancer. Material and Methods: We used our CRISPRa-competent PPKS (P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM)) model of adenocarcinoma to conduct targeted screening for drivers of resistance to MRTX133. Our library consisted of ∼450 frequently mutated and amplified putative oncogenes across human adenocarcinoma based on TCGA data. Mice were treated with 1e8 TU of lentiviral library through nasal instillation. After 6 weeks, treatment was initiated with 3 mg/kg MRTX1133 daily, 5 days/week (i.p.). Mice were sacrificed after 4 weeks of treatment and the guide distribution determined through bioinformatic analysis. Genes statistically significantly enriched (FDR<0.05) in the treatment group were determined. Cell lines with acquired resistance to MRTX1133 derived from the KPC PDAC model and the KPP lung adenocarcinoma (LUAD) model were generated using long-term, low-dose treatment. Resistance was confirmed with MTT drug response assays. In vitro combinatorial drug response was determined using a 72h, 96-well MTT assay, and clonogenic response using a 2 week, 384-well FACS assay. Mice were treated with 20 mg/kg MRTX1133 and/or 20mg/kg Temuterkib once daily, 7 days/week (i.p.) for 2 weeks. Results: We performed targeted in vivo CRISPRa screening for drivers of resistance to KRASi in an autochthons model of lung adenocarcinoma (LUAD) using a library targeting 452 genes commonly amplified or mutated in cancer. We identified a set of genes that were specifically enriched in at least 50% of tumors treated with MRTX1133, including MAPK1/ERK2. We also generated 16 cell lines with acquired resistance to MRTX1133, derived from KPC PDAC or KPP LUAD tumors. Transcriptomic analysis identified a large set of genes differentially expressed in cell with acquired resistance relative to controls. GSEA indicated that reactivation of the MAPK cascade as a common trait in resistant cells. To test if ERK inhibition synergizes with KRASi, we quantified the combinatorial response to MRTX1133 and Temuterkib, a specific inhibitor of ERK1 and ERK2, in a set of KPC and KPP derived cell lines, revealing significant synergy and reduced clonogenic potential in a majority of cell lines. A majority of cell lines with acquired resistance to MRTX1133 remained sensitive to Temuterkib. Finally, we tested combinatorial treatment of MRTX1133 and Temuterkib in a syngenetic subcutaneous model of PDAC, which showed at minimum additive effect. In conclusion we have identified ERK inhibition as a promising combination with KRAS inhibition in KRAS-mutant PDAC and LUAD. Citation Format: Fredrik I Thege, Amber Hoskins, Sonja M Woermann, Anirban Maitra. Identifying synergistic combinations with KRAS inhibition in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-15.

Genomic and transcriptomic analyses of a social hemipteran provide new insights into insect sociality.

The origin of sociality represents one of the most important evolutionary transitions. Insect sociality evolved in some hemipteran aphids, which can produce soldiers and normal nymphs with distinct morphology and behaviour through parthenogenesis. The lack of genomic data resources has hindered the investigations into molecular mechanisms underlying their social evolution. Herein, we generated the first chromosomal-level genome of a social hemipteran (Pseudoregma bambucicola) with highly specialized soldiers and performed comparative genomic and transcriptomic analyses to elucidate the molecular signatures and regulatory mechanisms of caste differentiation. P. bambucicola has a larger known aphid genome of 582.2 Mb with an N50 length of 11.24 Mb, and about 99.6% of the assembly was anchored to six chromosomes with a scaffold N50 of 98.27 Mb. A total of 14,027 protein-coding genes were predicted and 37.33% of the assembly were identified as repeat sequences. The social evolution is accompanied by a variety of changes in genome organization, including expansion of gene families related to transcription factors, transposable elements, as well as species-specific expansions of certain sugar transporters and UGPases involved in carbohydrate metabolism. We also characterized large candidate gene sets linked to caste differentiation and found evidence of expression regulation and positive selection acting on energy metabolism and muscle structure, explaining the soldier-specific traits including morphological and behavioural specialization, developmental arrest and infertility. Overall, this study offers new insights into the molecular basis of social aphids and the evolution of insect sociality and also provides valuable data resources for further comparative and functional studies.

EZH2 represses mesenchymal genes and upholds the epithelial state of breast carcinoma cells

Emerging studies support that the polycomb repressive complex 2 (PRC2) regulates phenotypic changes of carcinoma cells by modulating their shifts among metastable states within the epithelial and mesenchymal spectrum. This new role of PRC2 in cancer has been recently proposed to stem from the ability of its catalytic subunit EZH2 to bind and modulate the transcription of mesenchymal genes during epithelial-mesenchymal transition (EMT) in lung cancer cells. Here, we asked whether this mechanism is conserved in other types of carcinomas. By combining TGF-β-mediated reversible induction of epithelial to mesenchymal transition and inhibition of EZH2 methyltransferase activity, we demonstrate that EZH2 represses a large set of mesenchymal genes and favours the residence of breast cancer cells towards the more epithelial spectrum during EMT. In agreement, analysis of human patient samples supports that EZH2 is required to efficiently repress mesenchymal genes in breast cancer tumours. Our results indicate that PRC2 operates through similar mechanisms in breast and lung cancer cells. We propose that PRC2-mediated direct transcriptional modulation of the mesenchymal gene expression programme is a conserved molecular mechanism underlying cell dissemination across human carcinomas.

Change in prostate tissue gene expression following finasteride or doxazosin administration in the medical therapy for prostatic symptoms (MTOPS) study

Benign prostatic hyperplasia (BPH) may decrease patient quality of life and often leads to acute urinary retention and surgical intervention. While effective treatments are available, many BPH patients do not respond or develop resistance to treatment. To understand molecular determinants of clinical symptom persistence after initiating BPH treatment, we investigated gene expression profiles before and after treatments in the prostate transitional zone of 108 participants in the Medical Therapy of Prostatic Symptoms (MTOPS) Trial. Unsupervised clustering revealed molecular subgroups characterized by expression changes in a large set of genes associated with resistance to finasteride, a 5α-reductase inhibitor. Pathway analyses within this gene cluster found finasteride administration induced changes in fatty acid metabolism, amino acid metabolism, immune response, steroid hormone metabolism, and kinase activity within the transitional zone. We found that patients without this transcriptional response were highly likely to develop clinical progression, which is expected in 13.2% of finasteride-treated patients. Importantly, a patient’s transcriptional response to finasteride was associated with their pre-treatment kinase expression. Further, we identified novel expression signatures of finasteride resistance among the transcriptionally responded patients. These patients showed different gene expression profiles at baseline and increased prostate transitional zone volume compared to the patients who responded to the treatment. Our work suggests molecular mechanisms of clinical resistance to finasteride treatment that could be potentially helpful for personalized BPH treatment as well as new drug development to increase patient drug response.

A jasmonate-mediated regulatory network modulates diurnal floret opening time in rice.

Diurnal floret opening time (DFOT) is a pivotal trait for successful fertilization and hybrid breeding in rice. However, the molecular mechanism underlying this trait is poorly understood in rice. In this study, we combined the cytological, genetic and molecular studies to demonstrate that jasmonic acid (JA) regulates DFOT in rice through modulating the turgor and osmotic pressure of the lodicules. We show that lodicules undergo dramatic morphologic changes, accompanied by changes in water and sugar contents during the process of floret opening. Consistently, a large set of genes associated with cell osmolality and cell wall remodeling exhibits distinct expression profiles at different time points in our time-course transcriptomes of lodicules. Notably, a group of JA biosynthesis and signaling genes is continuously upregulated, accompanied by a gradual increase in JA accumulation as floret opening approaching. Furthermore, we demonstrate that the JA biosynthesis gene OsAOS1 is required for endogenous JA biosynthesis in lodicules and promoting rice DFOT. Moreover, OsMYC2, a master regulator of JA signaling, regulates rice DFOT by directly activating OsAOS1, OsSWEET4, OsPIP2;2 and OsXTH9. Collectively, our findings establish a core regulatory network mediated by JA for modulating rice DFOT and provide effective gene targets for the genetic improvement of DFOT in rice.

Cytokines and chemokines skin gene expression in correlation with immune cells in blood and severity in equine insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is the most frequent skin allergy of horses and is highly debilitating, especially in the chronic phase. IBH is caused by IgE-mediated hypersensitivity reactions to culicoides midge bites and an imbalanced immune response that reduces the welfare of affected horses. In the present study, we investigated the pathological mechanisms of IBH, aiming to understand the immune cell modulation in acute allergic skin lesions of IBH horses with the goal of finding possible biomarkers for a diagnostic approach to monitor treatment success. By qPCR, we quantified the gene expression of cytokines, chemokines, and immune receptors in skin punch biopsies of IBH with different severity levels and healthy horses simultaneously in tandem with the analysis of immune cell counts in the blood. Our data show an increase in blood eosinophils, monocytes, and basophils with a concomitant, significant increase in associated cytokine, chemokine, and immune cell receptor mRNA expression levels in the lesional skin of IBH horses. Moreover, IL-5Ra, CCR5, IFN-γ, and IL-31Ra were strongly associated with IBH severity, while IL-31 and IL-33 were rather associated with a milder form of IBH. In addition, our data show a strong correlation of basophil cell count in blood with IL-31Ra, IL-5, IL-5Ra, IFN-γ, HRH2, HRH4, CCR3, CCR5, IL-12b, IL-10, IL-1β, and CCL26 mRNA expression in skin punch biopsies of IBH horses. In summary, several cytokines and chemokines have been found to be associated with disease severity, hence contributing to IBH pathology. These molecules can be used as potential biomarkers to monitor the onset and progression of the disease or even to evaluate and monitor the efficacy of new therapeutic treatments for IBH skin allergy. To our knowledge, this is the first study that investigated immune cells together with a large set of genes related to their biological function, including correlation to disease severity, in a large cohort of healthy and IBH horses.

Circular cut codes in genetic information

In this work we present an analysis of the dinucleotide occurrences in the three codon sites 1–2, 2–3 and 1–3, based on a computation of the codon usage of three large sets of bacterial, archaeal and eukaryotic genes using the same method that identified a maximal C3 self-complementary trinucleotide circular code X in genes of bacteria and eukaryotes in 1996 (Arquès and Michel, 1996). Surprisingly, two dinucleotide circular codes are identified in the codon sites 1–2 and 2–3. Furthermore, these two codes are shifted versions of each other. Moreover, the dinucleotide code in the codon site 1–3 is circular, self-complementary and contained in the projection of X onto the 1st and 3rd bases, i.e. by cutting the middle base in each codon of X. We prove several results showing that the circularity and the self-complementarity of trinucleotide codes is induced by the circularity and the self-complementarity of its dinucleotide cut codes. Finally, we present several evolutionary approaches for an emergence of trinucleotide codes from dinucleotide codes.

Investigation of singlet-oxygen-responsive genes in the cyanobacterium Synechocystis PCC 6803.

Singlet oxygen (1O2) is an important reactive oxygen species whose formation by the type-II, light-dependent, photodynamic reaction is inevitable during photosynthetic processes. In the last decades, the recognition that 1O2 is not only a damaging agent, but can also affect gene expression and participates in signal transduction pathways has received increasing attention. However, contrary to several other taxa, 1O2-responsive genes have not been identified in the important cyanobacterial model organism Synechocystis PCC 6803. By using global transcript analysis we have identified a large set of Synechocystis genes, whose transcript levels were either enhanced or repressed in the presence of 1O2. Characteristic 1O2 responses were observed in several light-inducible genes of Synechocystis, especially in the hli (or scp) family encoding HLIP/SCP proteins involved in photoprotection. Other important 1O2-induced genes include components of the Photosystem II repair machinery (psbA2 and ftsH2, ftsH3), iron homeostasis genes isiA and idiA, the group 2 sigma factor sigD, some components of the transcriptomes induced by salt-, hyperosmotic and cold-stress, as well as several genes of unknown function. The most pronounced 1O2-induced upregulation was observed for the hliB and the co-transcribed lilA genes, whose deletion induced enhanced sensitivity against 1O2-mediated light damage. A bioreporter Synechocystis strain was created by fusing the hliB promoter to the bacterial luciferase (lux), which showed its utility for continuous monitoring of 1O2 concentrations inside the cell.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is aleading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

Natural genetic variation underlying the negative effect of elevated CO2 on ionome composition in Arabidopsis thaliana.

The elevation of atmospheric CO2 leads to a decline in plant mineral content, which might pose a significant threat to food security in coming decades. Although few genes have been identified for the negative effect of elevated CO2 on plant mineral composition, several studies suggest the existence of genetic factors. Here, we performed a large-scale study to explore genetic diversity of plant ionome responses to elevated CO2, using six hundred Arabidopsis thaliana accessions, representing geographical distributions ranging from worldwide to regional and local environments. We show that growth under elevated CO2 leads to a global decrease of ionome content, whatever the geographic distribution of the population. We observed a high range of genetic diversity, ranging from the most negative effect to resilience or even to a benefit in response to elevated CO2. Using genome-wide association mapping, we identified a large set of genes associated with this response, and we demonstrated that the function of one of these genes is involved in the negative effect of elevated CO2 on plant mineral composition. This resource will contribute to understand the mechanisms underlying the effect of elevated CO2 on plant mineral nutrition, and could help towards the development of crops adapted to a high-CO2 world.

Natural genetic variation underlying the negative effect of elevated CO2 on ionome composition in Arabidopsis thaliana

The elevation of atmospheric CO2 leads to a decline in plant mineral content, which might pose a significant threat to food security in coming decades. Although few genes have been identified for the negative effect of elevated CO2 on plant mineral composition, several studies suggest the existence of genetic factors. Here, we performed a large-scale study to explore genetic diversity of plant ionome responses to elevated CO2, using six hundred Arabidopsis thaliana accessions, representing geographical distributions ranging from worldwide to regional and local environments. We show that growth under elevated CO2 leads to a global decrease of ionome content, whatever the geographic distribution of the population. We observed a high range of genetic diversity, ranging from the most negative effect to resilience or even to a benefit in response to elevated CO2. Using genome-wide association mapping, we identified a large set of genes associated with this response, and we demonstrated that the function of one of these genes is involved in the negative effect of elevated CO2 on plant mineral composition. This resource will contribute to understand the mechanisms underlying the effect of elevated CO2 on plant mineral nutrition, and could help towards the development of crops adapted to a high-CO2 world.

Knock-out of BnHva22c reduces the susceptibility of Brassica napus to infection with the fungal pathogen Verticillium longisporum

Verticillium longisporum (Vl43) is a soilborne hemibiotrophic fungal pathogen causing stem striping on oilseed rape (OSR) and severe yield losses. Breeding for resistant varieties is the most promising approach to control this disease. Here, we report the identification of Hva22c as a novel susceptibility factor and its potential for improving OSR resistance. Hva22c is a member of the Hva22 gene family, originally described for barley (Hordeum vulgare). Several Hva22 members have been located at the endoplasmic reticulum. Hva22c is up-regulated in response to Vl43 in both Arabidopsis and OSR. We demonstrate that knock-out of Hva22c in OSR by CRISPR/Cas9 and its homolog in Arabidopsis by T-DNA insertion reduced plants’ susceptibility toVl43infection and impaired the development of disease symptoms. To understand the underlying mechanism, we analysed transcriptomic data from infected and non-infected roots of hva22c knock-out and wild type plants. We identified a homozygous mutant with frame-shifts in all four BnHva22c loci displaying a vastly altered transcriptional landscape at 6 dpi. Significantly, a large set of genes was suppressed under mock conditions including genes related to the endomembrane systems. Among the up-regulated genes we found several defense-related and phytohormone-responsive genes when comparing mutant to the wild type. These results demonstrate that Hva22c is functionally required for a fully compatible plant-fungus interaction. Its loss of function reduces plant susceptibility, most likely due to endoplasmatic reticulum and Golgi dysfunction accompanied by additionally activated defense responses. These findings can help improve OSR resistance to V. longisporum infection.

The nature and distribution of putative non-functional alleles suggest only two independent events at the origins of Astyanax mexicanus cavefish populations

BackgroundSeveral studies suggested that cavefish populations of Astyanax mexicanus settled during the Late Pleistocene. This implies that the cavefish’s most conspicuous phenotypic changes, blindness and depigmentation, and more cryptic characters important for cave life, evolved rapidly.ResultsUsing the published genomes of 47 Astyanax cavefish from la Cueva de El Pachón, El Sótano de la Tinaja, La Cueva Chica and El Sótano de Molino, we searched for putative loss-of-function mutations in previously defined sets of genes, i.e., vision, circadian clock and pigmentation genes. Putative non-functional alleles for four vision genes were identified. Then, we searched genome-wide for putative non-functional alleles in these four cave populations. Among 512 genes with segregating putative non-functional alleles in cavefish that are absent in surface fish, we found an enrichment in visual perception genes. Among cavefish populations, different levels of shared putative non-functional alleles were found. Using a subset of 12 genes for which putative loss-of-function mutations were found, we extend the analysis of shared pseudogenes to 11 cave populations. Using a subset of six genes for which putative loss-of-function mutations were found in the El Sótano del Toro population, where extensive hybridization with surface fish occurs, we found a correlation between the level of eye regression and the amount of putative non-functional alleles.ConclusionsWe confirm that very few putative non-functional alleles are present in a large set of vision genes, in accordance with the recent origin of Astyanax mexicanus cavefish. Furthermore, the genome-wide analysis indicates an enrichment of putative loss-of-function alleles in genes with vision-related GO-terms, suggesting that visual perception may be the function chiefly impacted by gene losses related to the shift from a surface to a cave environment.The geographic distribution of putative loss-of-function alleles newly suggests that cave populations from Sierra de Guatemala and Sierra de El Abra share a common origin, albeit followed by independent evolution for a long period. It also supports that populations from the Micos area have an independent origin. In El Sótano del Toro, the troglomorphic phenotype is maintained despite massive introgression of the surface genome.

HSPA9/mortalin inhibition disrupts erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies characterized by abnormal hematopoietic cell maturation, increased apoptosis of bone marrow cells, and anemia. They are the most common myeloid blood cancers in American adults. The full complement of gene mutations that contribute to the phenotypes or clinical symptoms in MDS is not fully understood. Around 10%-25% of MDS patients harbor an interstitial heterozygous deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes, including HSPA9. The HSPA9 gene encodes for the protein mortalin, a highly conserved heat shock protein predominantly localized in mitochondria. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+hematopoietic progenitor cells. In this study, we explored the role of HSPA9 in regulating erythroid maturation using human CD34+cells. We inhibited the expression of HSPA9 using gene knockdown and pharmacological inhibition and found that inhibition of HSPA9 disrupted erythroid maturation as well as increased expression of p53 in CD34+cells. To test whether the molecular mechanism of HSPA9 regulating erythroid maturation is TP53-dependent, we knocked downHSPA9 and TP53 individually or in combination in human CD34+cells. We found that the knockdown of TP53 partially rescued the erythroid maturation defect induced by HSPA9 knockdown, suggesting that the defect in cells with reduced HSPA9 expression is TP53-dependent. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to the anemia observed in del(5q)-associated MDS patients due to the activation of TP53.

Behavioral and transcriptomic analyses of mecp2 function in zebrafish.

Rett syndrome (RTT), a human neurodevelopmental disorder characterized by severe cognitive and motor impairments, is caused by dysfunction of the conserved transcriptional regulator Methyl-CpG-binding protein 2 (MECP2). Genetic analyses in mouse Mecp2 mutants, which exhibit key features of human RTT, have been essential for deciphering the mechanisms of MeCP2 function; nonetheless, our understanding of these complex mechanisms is incomplete. Zebrafish mecp2 mutants exhibit mild behavioral deficits but have not been analyzed in depth. Here, we combine transcriptomic and behavioral assays to assess baseline and stimulus-evoked motor responses and sensory filtering in zebrafish mecp2 mutants from 5 to 7 days post-fertilization (dpf). We show that zebrafish mecp2 function is required for normal thigmotaxis but is dispensable for gross movement, acoustic startle response, and sensory filtering (habituation and sensorimotor gating), and reveal a previously unknown role for mecp2 in behavioral responses to visual stimuli. RNA-seq analysis identified a large gene set that requires mecp2 function for correct transcription at 4 dpf, and pathway analysis revealed several pathways that require MeCP2 function in both zebrafish and mammals. These findings show that MeCP2's function as a transcriptional regulator is conserved across vertebrates and supports using zebrafish to complement mouse modeling in elucidating these conserved mechanisms.

Hypoxia drives shared and distinct transcriptomic changes in two invasive glioma stem cell lines

Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.

Identification of a chromatin-bound ERRα interactome network in mouse liver

ObjectivesEstrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver. MethodsRIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes. ResultsCharacterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their colocalization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration. ConclusionsOur work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs.

Abstract 148: Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % of patients. Recent results show that relapse is due to a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. Thus, developing a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution is crucial. In this study, we used whole exome, barcode and RNA sequencing to characterize the landscape of genomic alterations and also track and identify the clonal subpopulations, clonal and genetic drivers of lungs metastasis. Methods: We injected barcoded PDX (OS17) cells intravenously in ten SCID mice to track the clonal subpopulation of cells that metastasize to the lungs. Metastasized lung nodules were collected following death or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -80°C. DNA and RNA was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Four (40%) of the mice developed lung metastases with three mice having multiple metastatic lung nodules. Surgery to remove the metastatic nodules was performed 216 ± 55.5 days following tumor cell injection. Nine metastatic lung nodules from three mice were used for sequencing and analysis. A Shannon-Weaver and Jaccard similarity index show a diversity in clonal architecture between the lung nodules. A total of 15,394 somatic variants was identified including 12,547 single nucleotide variants (SNV), 4054 synonymous, 2662 missense, 28 nonsense mutations and 1639 somatic indels. The SNVs accounted for 81% of all variants followed by CNV 11 % and indels 8% respectively. Synonymous and missense mutations were the most frequently observed in all samples. Copy number loss was two-fold compared to copy number gain across all tumors. Furthermore, the deletions span longer genomic distances compared to amplifications. The mutational spectrum showed that the highest substitutions were C>T/G>A followed by T>C/A>G; the least substitutions were T>A/A>T. Also, we identified a large set of genes with genomic aberrations. These include amplifications in VEGF, RB1, RUNX, PARP4, ICAM3, EGFR, BRCA2, COLA6A1, COLA6A2, CCND3, CDKN2D etc. Conclusions: In the current model, we identified multiple tumorigenic seeding clones and the diverse set of genes mutated in osteosarcoma. Further analysis of the mutational signature resulted in the identification of potential genes that act as drivers in the lung metastatic process. Acknowledgments: Swim Across America, the Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Wendong Zhang, Zhongting Zhang, Xu Zhaohui, Sankaranarayanan Kannan, Yifei Wang, Zhou Xin, Yi Yanhua, Michael Roth, Jonathan Gill, Richard Gorlick. Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model [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 148.

Noradrenaline release from the locus coeruleus shapes stress-induced hippocampal gene expression.

Exposure to an acute stressor triggers a complex cascade of neurochemical events in the brain. However, deciphering their individual impact on stress-induced molecular changes remains a major challenge. Here, we combine RNA sequencing with selective pharmacological, chemogenetic, and optogenetic manipulations to isolate the contribution of the locus coeruleus-noradrenaline (LC-NA) system to the acute stress response in mice. We reveal that NA release during stress exposure regulates a large and reproducible set of genes in the dorsal and ventral hippocampus via β-adrenergic receptors. For a smaller subset of these genes, we show that NA release triggered by LC stimulation is sufficient to mimic the stress-induced transcriptional response. We observe these effects in both sexes, and independent of the pattern and frequency of LC activation. Using a retrograde optogenetic approach, we demonstrate that hippocampus-projecting LC neurons directly regulate hippocampal gene expression. Overall, a highly selective set of astrocyte-enriched genes emerges as key targets of LC-NA activation, most prominently several subunits of protein phosphatase 1 (Ppp1r3c, Ppp1r3d, Ppp1r3g) and type II iodothyronine deiodinase (Dio2). These results highlight the importance of astrocytic energy metabolism and thyroid hormone signaling in LC-mediated hippocampal function and offer new molecular targets for understanding how NA impacts brain function in health and disease.