Induction Of Early Genes Research Articles

Endothelial Beta 1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgb1) subunit is highly expressed in endothelial cells (EC). EC itgb1 is necessary for the formation of capillary networks during embryonic during development and its knockdown blunts the reactive hyperemia that manifests during ischemia reperfusion. We investigated the contribution of EC itgb1 in microcirculatory function and glucose uptake with emphasis in skeletal muscle. We hypothesized that loss of EC itgb1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. Mice with (itgb1fl/flSCLcre) and without (itgb1fl/fl) tamoxifen inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction had comparable exercise tolerance and pulmonary and cardiac functions. Using robust in vivo experimental platforms (i.e., intravital microscopy and hyperinsulinemic-euglycemic clamp), we show that itgb1fl/flSCLcre mice compared to itgb1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgb1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Endothelial β1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (EC). EC itgβ1 is necessary for the formation of capillary networks during embryonic during development and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study we investigated the contribution of skeletal muscle EC itgβ1 in microcirculatory function and glucose uptake. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. We found that mice with (itgβ1fl/flSCLcre) and without (itgβ1fl/fl) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1fl/flSCLcre mice compared to itgβ1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both invivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an invivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Transcriptional activation of candidate paused genes after partial hepatectomy.

Partial hepatectomy is a model of acute liver injury that is known to induce a strong reprogrammation of gene expression. Transcriptional induction of Immediate Early Genes is extremely fast and this would be due to the release of RNA Polymerase II poised for elongation at 'paused' genes. Using bioinformatic analysis, we identified 23 genes sharing features of paused genes before hepatectomy, and with predicted quick and strong expression induction after. This transcriptional dynamic, confirmed by RT-qPCR for Jun , Fos , Btg2, is very precocious. RNA Pol II CTD Ser2 hyperphosphorylation indicates a switch to productive elongation and release from transcriptional pause.

PUB63, A Novel Early Heat Responsive Ubiquitin E3 Ligase from Rice Implicates Heat Stress Tolerance

Heat stress has emerged as a major environmental challenge triggering reduced crop yields, compromised growth, and negatively affecting the development of crops. The cellular response to heat stress involves transcriptional and translational reprogramming and regulation of protein homeostasis which is crucial for the heat stress response and leads to the differential expression of many genes associated with protein turnover mechanisms. Plant U-box (PUB) proteins are a class of the Ubiquitin-E3 ligase involved in the Ubiquitin-proteasome pathway. In the present study, we investigated the role of a PUB protein from rice, OsPUB63, involved in heat stress responses in rice. We also identified all PUB coding genes from O. sativa indica and compared with O. sativa japonica. A quantitative real time PCR demonstrated, OsPUB63 as an early heat inducible gene in rice and the subcellular localization study confirmed its presence in the nucleus and cytosol. OsPUB63 was demonstrated as a TPR repeat containing protein with molecular weight of 39.3 KDa. Using a combination of genetic, biochemical, and physiological assays, our work establishes OsPUB63 as crucial in regulating the heat stress response in rice. Specifically, we show that OsPUB63 enhances the heat stress tolerance in transgenic Arabidopsis by promoting the accumulation of proline and reduced MDA content. Our work has identified PUB63 as the first PUB gene family E3 ligase from rice which is implicated in heat stress response and provide a crucial target for improving crop yields under high-temperature conditions in rice.

Acute stress modulates the outcome of traumatic brain injury-associated gene expression and behavioral responses.

Psychological stress and traumatic brain injury (TBI) result in long-lasting emotional and behavioral impairments in patients. So far, the interaction of psychological stress with TBI not only in the brain but also in peripheral organs is poorly understood. Herein, the impact of acute stress (AS) occurring immediately before TBI is investigated. For this, a mouse model of restraint stress and TBI was employed, and their influence on behavior and gene expression in brain regions, the hypothalamic-pituitary-adrenal (HPA) axis, and peripheral organs was analyzed. Results demonstrate that, compared to single AS or TBI exposure, mice treated with AS prior to TBI showed sex-specific alterations in body weight, memory function, and locomotion. The induction of immediate early genes (IEGs, e.g., c-Fos) by TBI was modulated by previous AS in several brain regions. Furthermore, IEG upregulation along the HPA axis (e.g., pituitary, adrenal glands) and other peripheral organs (e.g., heart) was modulated by AS-TBI interaction. Proteomics of plasma samples revealed proteins potentially mediating this interaction. Finally, the deletion of Atf3 diminished the TBI-induced induction of IEGs in peripheral organs but left them largely unaltered in the brain. In summary, AS immediately before brain injury affects the brain and, to a strong degree, also responses in peripheral organs.

CREB Is Activated by the SCF/KIT Axis in a Partially ERK-Dependent Manner and Orchestrates Survival and the Induction of Immediate Early Genes in Human Skin Mast Cells

cAMP response element binding protein (CREB) functions as a prototypical stimulus-inducible transcription factor (TF) that initiates multiple cellular changes in response to activation. Despite pronounced expression in mast cells (MCs), CREB function is surprisingly ill-defined in the lineage. Skin MCs (skMCs) are critical effector cells in acute allergic and pseudo-allergic settings, and they contribute to various chronic dermatoses such as urticaria, atopic dermatitis, allergic contact dermatitis, psoriasis, prurigo, rosacea and others. Using MCs of skin origin, we demonstrate herein that CREB is rapidly phosphorylated on serine-133 upon SCF-mediated KIT dimerization. Phosphorylation initiated by the SCF/KIT axis required intrinsic KIT kinase activity and partially depended on ERK1/2, but not on other kinases such as p38, JNK, PI3K or PKA. CREB was constitutively nuclear, where phosphorylation occurred. Interestingly, ERK did not translocate to the nucleus upon SCF activation of skMCs, but a fraction was present in the nucleus at baseline, and phosphorylation was prompted in the cytoplasm and nucleus in situ. CREB was required for SCF-facilitated survival, as demonstrated with the CREB-selective inhibitor 666-15. Knock-down of CREB by RNA interference duplicated CREB's anti-apoptotic function. On comparison with other modules (PI3K, p38 and MEK/ERK), CREB was equal or more potent at survival promotion. SCF efficiently induces immediate early genes (IEGs) in skMCs (FOS, JUNB and NR4A2). We now demonstrate that CREB is an essential partaker in this induction. Collectively, the ancient TF CREB is a crucial component of skMCs, where it operates as an effector of the SCF/KIT axis, orchestrating IEG induction and lifespan.

Social regulation of immediate early gene induction in gonadotropin releasing-hormone 1 neurons and singing behavior in canaries (Serinus canaria)

Social cues modulate the neuroendocrine control of reproduction. However, the neural systems involved in the integration of social cues are not well described. Gonadotropin-releasing hormone 1 (GnRH1) cells in the preoptic area (POA) are the final common node that links the brain with peripheral reproductive physiology. These experiments investigated whether induction of the immediate early gene, EGR1, in anatomically localized GnRH1 cell populations in Border canaries is regulated by the social environment. First, we characterized behavioral modifications in singing behavior and found males paired with a female for 2 weeks significantly reduced many aspects of singing behavior. However, paired males had a significantly higher percentage of GnRH1 cells co-labeled with EGR1. The second experiment manipulated the social environment by pairing males and females in mixed sex dyads, same sex dyads or housed birds in isolation. Only when birds are paired in mixed sex dyads was there a significantly greater percentage of GnRH1 cells expressing EGR1 cells. Increased GnRH1-EGR1 co-expression was localized to the rostral POA. These data reveal that discrete GnRH1 cells are involved in the neural integration of specific social cues and support the hypothesis that the POA exhibits functional topography related to courtship and sexual behaviors.

Analysis of Growth Phases of Enterotoxigenic Escherichia coli Reveals a Distinct Transition Phase before Entry into Early Stationary Phase with Shifts in Tryptophan, Fucose, and Putrescine Metabolism and Degradation of Neurotransmitter Precursors.

ABSTRACTEnterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to early stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition from logarithmic to stationary growth. The transient phase was found to be different from the subsequent induction of early stationary phase-induced genes. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence from mid-logarithmic to the start of the stationary phase. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., l-tyrosine, l-tryptophan, l-phenylalanine, l-leucine, and l-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire combined transcriptome and metabolome revealed that significant pathways during progression from logarithmic to early stationary phase are involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli.IMPORTANCE We show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of certain amino acids. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described “indole peak,” rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolome is short-lived and disappears when bacteria enter the early stationary phase. We suggest that these changes mainly prepare bacteria for ceased growth, but based on the pathways involved, we could suggest that this transient phase substantially influences survival and virulence.

ERK-dependent induction of the immediate-early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs-GPCR signaling for neuritogenesis.

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50,upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis.

Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

IntroductionCysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated.HypothesisWe hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells.Methods and ResultsPC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction.ConclusionThe results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

Capturing an Early Gene Induction Event during Wood Decay by the Brown Rot Fungus Rhodonia placenta.

ABSTRACTBrown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step itself might require induction. To capture this early gene regulation event, here, we integrated fine-scale cryosectioning with whole-transcriptome sequencing to dissect gene expression at the single-hyphal-cell scale (tens of micrometers). This improved the spatial resolution 50-fold, relative to previous work, and we were able to capture the activity of the first 100 μm of hyphal front growth by Rhodonia placenta in aspen wood. This early decay period was dominated by delayed gene expression patterns as the fungus ramped up its mechanism. These delayed DEGs included many genes implicated in ROS pathways (lignocellulose oxidation [LOX]) that were previously and incorrectly assumed to be constitutively expressed. These delayed DEGs, which include those with and without predicted functions, also create a focused subset of target genes for functional genomics. However, this delayed pattern was not universal, with a few genes being upregulated immediately at the hyphal front. Most notably, this included a gene commonly implicated in hydroquinone and iron redox cycling: benzoquinone reductase.IMPORTANCE Earth’s aboveground terrestrial biomass is primarily wood, and fungi dominate wood decomposition. Here, we studied these fungal pathways in a common “brown rot”-type fungus, Rhodonia placenta, that selectively extracts sugars from carbohydrates embedded within wood lignin. Using a space-for-time design to map fungal gene expression at the extreme hyphal front in wood, we made two discoveries. First, we found that many genes long assumed to be “on” (constitutively expressed) from the very beginning of decay were instead “off” before being upregulated, when mapped (via transcriptome sequencing [RNA-seq]) at a high resolution. Second, we found that the gene encoding benzoquinone reductase was “on” in incipient decay and quickly downregulated, implying a key role in “kick-starting” brown rot.

Identification and validation of the wound and insect bite early inducible promoter from Arabidopsis thaliana.

The online version contains supplementary material available at 10.1007/s13205-022-03143-0.

MSK-Mediated Phosphorylation of Histone H3 Ser28 Couples MAPK Signalling with Early Gene Induction and Cardiac Hypertrophy.

Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy.

Assessing Gq-GPCR-induced human astrocyte reactivity using bioengineered neural organoids.

Astrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor-driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing.

FGF7 Supports Hematopoietic Stem and Progenitor Cells and Niche-Dependent Myeloblastoma Cells Via Autocrine Action On Bone Marrow Stromal Cells In Vitro

Some fibroblast growth factor (FGF)/FGF receptor (FGFR) systems, namely FGF1 and FGF2 which are produced mainly by megakaryocytes, and their cognate receptors expressed on either hematopoietic stem or progenitor cells (HSPCs) or bone marrow (BM) stromal cells, are reported to support HSPCs under stressed conditions. In this study, we show that mesenchymal cell-derived FGF7 (also called keratinocyte growth factor or KGF) may be a novel niche factor for HSPC support and niche-dependent leukemia-initiating cell growth. We previously reported that mouse embryonic fibroblasts (MEFs) have characteristics that mimic BM stromal cells and possess an ability of HSPCs support in vitro, and that this ability is profoundly attenuated in MEFs deficient for the MED1 subunit of the Mediator transcriptional master coregulator complex, subcomplex of RNA polymerase II holoenzyme. We found through the microarray analyses that the FGF7 expression (both at mRNA and protein levels) was specifically and profoundly attenuated in Med1-/- MEFs compared with Med1+/+ MEFs. When normal mouse BM cells were cocultured with mitomycin C-treated Med1+/+ MEFs or BM stromal cells (OP-9 or MS-5) in the presence of anti-FGF7 antibody, the DNA synthesis and resultant growth of BM cells, and the number of long-term culture-initiating cells (LTC-ICs), were significantly suppressed. However, the addition of anti-FGF7 antibody did not enhance cell death in BM cells. In contrast, the addition of recombinant FGF7 to the coculture of BM cells with mitomycin C-treated Med1-/- MEFs robustly upregulated the DNA synthesis and growth of BM cells, and the number of LTC-ICs. The Ph+ myeloblastoma cells MB-1, originally isolated from a patient with blast crisis chronic myeloid leukemia, is niche-dependent myeloblastic cells that require mesenchymal stromal cells for survival and growth. During coculture typically with OP-9 BM stromal cells, these MB-1 cells form cobblestone areas, which most probably reconstitute a stochastic model of leukemic stem or initiating cells in vitro. We next analyzed the role of FGF7 in support of MB-1 cells for growth and survival during coculture. Indeed, the addition of anti-FGF7 antibody to the coculture with mitomycin C-treated BM stromal cells (either OP-9 or MS-5) substantially attenuated DNA synthesis and resultant proliferation of stromal cell-dependent myeloblastoma cells MB1. Notably, cobblestone formation (i.e., signature of stem cell phenotype) of MB-1 cells was significantly attenuated when anti-FGF7 antibody was added to the coculture with OP-9 cells. However, the addition of anti-FGF7 antibody did not elicit cell death of MB-1 cells. Thus, FGF7 appears to promote both proliferation and maintenance of leukemia stem or initiating cell feature of MB-1 cells. While neither FGF7 nor its cognate receptor FGFR2IIIb mRNAs were detectable in BM cells, the mesenchymal cells including Med1+/+ MEFs and (OP-9 and MS-5) BM stromal cells expressed them. The expression levels of FGFR2IIIb were comparable between Med1+/+ and Med1-/- MEFs, and BM stromal cells. Therefore, we hypothesized that FGFR2IIIb on these mesenchymal stromal cells might be functional and possibly elicit secretion of niche signal(s) secondarily in response to the downstream target gene(s) of FGFR2IIIb, followed by subsequent support of HSPCs and leukemic cells. To prove that FGF7 is functional in these cells, we examined response of the downstream targets of FGFR2IIIb in these cells, including phosphorylation of FRS2 (the immediate downstream target of FGFR2IIIb tyrosine kinase), phosphorylation of MAP kinases (the intermediate hub of various intracellular signals), and mRNA expression of immediate early response genes including c-Jun, c-Fos and c-Myc. Indeed, exogenously added recombinant FGF7 elicited phosphorylation of both FRS2 and MAP kinases within 10 min, and full induction of these immediate early response genes within 30 min, similarly in both Med1+/+ and Med1-/- MEFs. The OP-9 and MS-5 BM stromal cells also exhibited similar responses to exogenously added FGF7. These results indicate that FGFR2IIIb expressed on mesenchymal stromal cells are functional, and that FGF7 and FGFR2IIIb produced by these cells constitute an autocrine (or paracrine) loop. Taken together, we propose that FGF7 produced by BM stromal cells supports HSPCs and leukemic progenitor cells indirectly via FGFR2IIIb expressed on the BM stromal cells. Disclosures:No relevant conflicts of interest to declare.

Profiling DNA break sites and transcriptional changes in response to contextual fear learning.

Neuronal activity generates DNA double-strand breaks (DSBs) at specific loci in vitro and this facilitates the rapid transcriptional induction of early response genes (ERGs). Physiological neuronal activity, including exposure of mice to learning behaviors, also cause the formation of DSBs, yet the distribution of these breaks and their relation to brain function remains unclear. Here, following contextual fear conditioning (CFC) in mice, we profiled the locations of DSBs genome-wide in the medial prefrontal cortex and hippocampus using γH2AX ChIP-Seq. Remarkably, we found that DSB formation is widespread in the brain compared to cultured primary neurons and they are predominately involved in synaptic processes. We observed increased DNA breaks at genes induced by CFC in neuronal and non-neuronal nuclei. Activity-regulated and proteostasis-related transcription factors appear to govern some of these gene expression changes across cell types. Finally, we find that glia but not neurons have a robust transcriptional response to glucocorticoids, and many of these genes are sites of DSBs. Our results indicate that learning behaviors cause widespread DSB formation in the brain that are associated with experience-driven transcriptional changes across both neuronal and glial cells.

Social Experience Interacts with Serotonin to Affect Functional Connectivity in the Social Behavior Network following Playback of Social Vocalizations in Mice.

Past social experience affects the circuitry responsible for producing and interpreting current behaviors. The social behavior network (SBN) is a candidate neural ensemble to investigate the consequences of early-life social isolation. The SBN interprets and produces social behaviors, such as vocalizations, through coordinated patterns of activity (functional connectivity) between its multiple nuclei. However, the SBN is relatively unexplored with respect to murine vocal processing. The serotonergic system is sensitive to past experience and innervates many nodes of the SBN; therefore, we tested whether serotonin signaling interacts with social experience to affect patterns of immediate early gene (IEG; cFos) induction in the male SBN following playback of social vocalizations. Male mice were separated into either social housing of three mice per cage or into isolated housing at 18–24 d postnatal. After 28–30 d in housing treatment, mice were parsed into one of three drug treatment groups: control, fenfluramine (FEN; increases available serotonin), or pCPA (depletes available serotonin) and exposed to a 60-min playback of female broadband vocalizations (BBVs). FEN generally increased the number of cFos-immunoreactive (-ir) neurons within the SBN, but effects were more pronounced in socially isolated mice. Despite a generalized increase in cFos immunoreactivity, isolated mice had reduced functional connectivity, clustering, and modularity compared with socially reared mice. These results are analogous to observations of functional dysconnectivity in persons with psychopathologies and suggests that early-life social isolation modulates serotonergic regulation of social networks.

LGG-14. MULTI-OMIC ANALYSIS OF MAPK ACTIVATION IN PEDIATRIC PILOCYTIC ASTROCYTOMA

Pilocytic astrocytomas (PA) are low-grade gliomas (pLGG) and are the most frequent childhood brain tumors. They are characterized by oncogene-induced senescence (OIS) initiated and sustained by senescence-associated secretory phenotype (SASP) factors. OIS and SASP in PA are thought to be driven by aberrations of the mitogen-activated protein kinase (MAPK) pathway (e.g. KIAA1549:BRAF fusion, BRAFV600E mutation, for the most common MAPK alterations occuring in PA), leading to its sustained activation. The MAPK pathway cascade is activated in a sequential manner: 1) ERK activation, which phosphorylates downstream partners in both cytoplasm and nucleus. 2) ERK-mediated induction of immediate early genes encoding transcription factors. 3) Induction of MAPK target genes expression. 4) Activation of downstream pathways. Our aim is to unravel the molecular partners involved at each level of the sustained MAPK pathway activation in pLGG with different genetic backgrounds (KIAA1549:BRAF fusion and BRAFV600E mutation), and leading to the induction of OIS and SASP factors expression. pLGG cell lines DKFZ-BT66 (KIAA1549:BRAF) and BT-40 (BRAFV600E) were treated with the MEK inhibitor trametinib at key time points, and gene expression profile analysis was performed, allowing transcriptome analysis at each step of the MAPK cascade. This will be combined with a whole proteomic and phospho-proteomic analysis. Combination of the transcriptome and proteome data layers will allow the identification of a) downstream targetable partners activated by the MAPK pathway involved in PA senescence, b) new putative targets that might bring benefit in combination with MAPK inhibitors.

Regulation of dopamine-dependent transcription and cocaine action by Gadd45b

Exposure to drugs of abuse produces robust transcriptional and epigenetic reorganization within brain reward circuits that outlives the direct effects of the drug and may contribute to addiction. DNA methylation is a covalent epigenetic modification that is altered following stimulant exposure and is critical for behavioral and physiological adaptations to drugs of abuse. Although activity-related loss of DNA methylation requires the Gadd45 (Growth arrest and DNA-damage-inducible) gene family, very little is known about how this family regulates activity within the nucleus accumbens or behavioral responses to drugs of abuse. Here, we combined genome-wide transcriptional profiling, pharmacological manipulations, electrophysiological measurements, and CRISPR tools with traditional knockout and behavioral approaches in rodent model systems to dissect the role of Gadd45b in dopamine-dependent epigenetic regulation and cocaine reward. We show that acute cocaine administration induces rapid upregulation of Gadd45b mRNA in the rat nucleus accumbens, and that knockout or site-specific CRISPR/Cas9 gene knockdown of Gadd45b blocks cocaine conditioned place preference. In vitro, dopamine treatment in primary striatal neurons increases Gadd45b mRNA expression through a dopamine receptor type 1 (DRD1)-dependent mechanism. Moreover, shRNA-induced Gadd45b knockdown decreases expression of genes involved in psychostimulant addiction, blocks induction of immediate early genes by DRD1 stimulation, and prevents DRD1-mediated changes in DNA methylation. Finally, we demonstrate that Gadd45b knockdown decreases striatal neuron action potential burst duration in vitro, without altering other electrophysiological characteristics. These results suggest that striatal Gadd45b functions as a dopamine-induced gene that is necessary for cocaine reward memory and DRD1-mediated transcriptional activity.