Activation Of Response Genes Research Articles

Bortezomib Stabilizes and Activates p53 in Proliferative Compartments of Both Normal and Tumor Tissues In Vivo

p53 protein, activated and stabilized by posttranslational modifications, performs its major functions by inducing DNA repair, cell-cycle arrest, or apoptosis through transcriptional activation. Here, we determined the ability of p53 protein stabilized via proteasome inhibition to perform similar functions as p53 induced by stresses such as DNA damage. Treating mice with the proteasome inhibitor bortezomib stabilized p53 in stem/progenitor cells of the intestine and stomach, in other proliferating tissues, and in intestinal tumors. Robust basal p53 mRNA levels were observed in the same compartments where p53 was stabilized. Spatial activation of p53 target genes in response to bortezomib in the small intestine demonstrated that CDKN1A and BAX were upregulated in the proliferative crypts but not in the differentiated villi of the small intestine; PUMA was specifically activated at the crypt base of p53 wild-type mice. Thus, cellular context determines the p53 transcriptional target selection. p53-dependent apoptosis was induced in Lgr5-expressing stem cells of the small intestine and high p53 transcriptional activity and apoptosis was induced in intestinal adenomas and in xenograft tumors. Bortezomib inhibited the growth of intestinal adenomas and xenograft tumors with wild-type p53, indicating the importance of p53 in the response to proteasome inhibitors in tissue homeostasis and in cancer therapy. SIGNIFICANCE: These findings show that bortezomib is less active in p53-defective tumors, yet its success in treating multiple myeloma suggests its use can be extended to p53-proficient solid tumors.

The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair

Cytokine tumor necrosis factor (TNF)-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. We performed transcriptomic and epigenomic analysis of the TNF response in primary human macrophages and revealed late-phase activation of SREBP2, the master regulator of cholesterol biosynthesis genes. TNF stimulation extended the genomic profile of SREBP2 occupancy to include binding to and activation of inflammatory and interferon response genes independently of its functions in sterol metabolism. Genetic ablation of SREBP function shifted the balance of macrophage polarization from an inflammatory to a reparative phenotype in peritonitis and skin wound healing models. Genetic ablation of SREBP activity in myeloid cells or topical pharmacological inhibition of SREBP improved skin wound healing under homeostatic and chronic inflammatory conditions. Our results identify a function and mechanism of action for SREBPs in augmenting TNF-induced macrophage activation and inflammation and open therapeutic avenues for promoting wound repair.

The role of transposable elements in the ecological morphogenesis under the influence of stress

In natural selection, insertional mutagenesis is an important source of genome variability. Transposons are sensors of environmental stress effects, which contribute to adaptation and speciation. These effects are due to changes in the mechanisms of morphogenesis, since transposons contain regulatory sequences that have cis and trans effects on specific protein-coding genes. In variability of genomes, the horizontal transfer of transposons plays an important role, because it contributes to changing the composition of transposons and the acquisition of new properties. Transposons are capable of site-specific transpositions, which lead to the activation of stress response genes. Transposons are sources of non-coding RNA, transcription factors binding sites and protein-coding genes due to domestication, exonization, and duplication. These genes contain nucleotide sequences that interact with non-coding RNAs processed from transposons transcripts, and therefore they are under the control of epigenetic regulatory networks involving transposons. Therefore, inherited features of the location and composition of transposons, along with a change in the phenotype, play an important role in the characteristics of responding to a variety of environmental stressors. This is the basis for the selection and survival of organisms with a specific composition and arrangement of transposons that contribute to adaptation under certain environmental conditions. In evolution, the capability to transpose into specific genome sites, regulate gene expression, and interact with transcription factors, along with the ability to respond to stressors, is the basis for rapid variability and speciation by altering the regulation of ontogenesis. The review presents evidence of tissue-specific and stage-specific features of transposon activation and their role in the regulation of cell differentiation to confirm their role in ecological morphogenesis.

Reduced PAX2 expression in murine fallopian tube cells enhances estrogen receptor signaling

High-grade serous ovarian cancer (HGSOC) is thought to progress from a series of precursor lesions in the fallopian tube epithelium (FTE). One of the preneoplastic lesions found in the FTE is called a secretory cell outgrowth (SCOUT), which is partially defined by a loss of paired box 2 (PAX2). In the present study, we developed PAX2-deficient murine cell lines in order to model a SCOUT and to explore the role of PAX2 loss in the etiology of HGSOC. Loss of PAX2 alone in the murine oviductal epithelium (MOE) did not induce changes in proliferation, migration and survival in hypoxia or contribute to resistance to first line therapies, such as cisplatin or paclitaxel. RNA sequencing of MOE PAX2shRNA cells revealed significant alterations in the transcriptome. Silencing of PAX2 in MOE cells produced a messenger RNA expression pattern that recapitulated several aspects of the transcriptome of previously characterized human SCOUTs. RNA-seq analysis and subsequent qPCR validation of this SCOUT model revealed an enrichment of genes involved in estrogen signaling and an increase in expression of estrogen receptor α. MOE PAX2shRNA cells had higher estrogen signaling activity and higher expression of putative estrogen responsive genes both in the presence and absence of exogenous estrogen. In summary, loss of PAX2 in MOE cells is sufficient to transcriptionally recapitulate a human SCOUT, and this model revealed an enrichment of estrogen signaling as a possible route for tumor progression of precursor lesions in the fallopian tube.

Abstract 3479: Functional significance of ESR1 fusions with diverse gene partners in endocrine therapy resistant breast cancer

Abstract Emerging evidence suggests that ESR1 fusion transcripts that produce functional fusion proteins in estrogen receptor positive (ER+) breast cancer play a role in acquired endocrine therapy resistance. We recently reported two in-frame ESR1 fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, identified in patients with endocrine therapy resistant breast cancer. Both ESR1 fusions generated stable in-frame, fusion proteins that were transcriptionally active, driving endocrine therapy resistant proliferation and also promoting an epithelial-to-mesenchymal transition (EMT) transcriptional program leading to metastasis in experimental models. Here, we extend our studies by examining properties of additional ESR1 fusions with diverse partner genes, ESR1-DAB2, ESR1-GYG1, ESR1-SOX9, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B, all identified in endocrine-refractory breast tumors, to better understand the role of ESR1fusion gene formation in endocrine therapy resistance. Structurally, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B fusions retain the first 6 exons of ESR1, therefore lacking ESR1 exons encoding the ligand-binding domain (LBD) that endocrine therapies recognize, but are instead fused in-frame to C-terminal sequences from partner genes, which follows the same fusion pattern of previously identified ESR1-YAP1, ESR1-PCDH11X, ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 fusions. ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B constructs, along with ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 constructs were stably expressed in an ER+ breast cancer cell line, T47D, and all produced stable fusion proteins. ESR1-ARNT2 and ESR1-SOX9 promoted hormone-independent and fulvestrant-resistant cell proliferation that was sensitive to cyclin-dependent kinase (CDK) 4/6 inhibitors, palbociclib and abemaciclib. In addition, ESR1-ARNT2 induced hormone-independent activation of estrogen responsive genes (TFF1, GREB1, and PGR), and EMT genes (SNAI1 and VCAN) along with upregulation of Snail protein. These results indicate that ESR1-ARNT2 and ESR1-SOX9 have similar functional and pharmacological properties to our previously described ESR1-YAP1 and ESR1-PCDH11X fusions. These data further support a role for ESR1 fusions in driving not only endocrine therapy resistance but also activating the metastatic process. Ongoing studies are examining the ability of ESR1 fusions to induce EMT phenotypes and to determine structure function relationships of ESR1 fusions. Although the formation of ESR1 fusions likely confers resistance to all endocrine therapies that target the LBD, ESR1 fusion driven growth remained sensitive to CDK4/6 inhibitor treatment therefore providing rationale for testing ESR1 fusion detection in guiding treatment with CDK4/6 monotherapy. Citation Format: Jonathan T. Lei, Xuxu Gou, Sinem Seker, Vaishnaivi Devorakonda, Kimberly R. Holloway, Adrian V. Lee, Dan R. Robinson, Matthew J. Ellis. Functional significance of ESR1 fusions with diverse gene partners in endocrine therapy resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3479.

Abstract PL02-04: Genomic and epigenomic studies of cholangiocarcinoma in diverse populations

Abstract It is well-known that Asian prevalent cancers are often associated with a pathogen or carcinogen. For example, hepatocellular carcinoma is driven by hepatitis B and C viruses, gastric cancer by Helicobacter pylori, and nasopharyngeal cancer by Epstein-Barr virus. In this presentation, we focus on biliary tract cancer or cholangiocarcinoma (CCA) of different etiologies which are associated with chronic carcinogenic exposures leading to distinct genomic and epigenomic changes. CCA is a malignant tumor of bile duct epithelial cells with very poor prognosis which accounted for approximately 5% of all primary liver cancers diagnosed worldwide. It is broadly divided into intrahepatic, perihilar, and distal CCAs which may differ in their spectrum and frequency of genetic alterations, possibly reflecting differences in cell of origin and tumorigenic process. CCA displays distinct molecular mechanisms that require different therapeutic regimes for effective treatment representing an important unmet clinical need. Similarly, the genomic and epigenomic changes may serve as effective biomarkers for disease detection and monitoring. Risk factors for CCA are related to geography and etiology. In Western countries, primary sclerosing cholangitis (PSC) is the most common risk factor of CCA, and other risk factors include hepatolithiasis, choledochal cysts, cirrhosis, diabetes, obesity, alcohol consumption and smoking. On the other hand, in the northeast region of Thailand, Laos and Cambodia, infestation of liver fluke called Opisthorchis viverrini (OV) has been associated with the highest incidence of CCA, with approximately 20,000 cases annually. Once ingested, OV can inhabit the biliary tract within the human host for over 10 years, promoting carcinogenesis potentially via 1) mechanical damage to the biliary epithelia caused by the feeding activities of the parasites, 2) immunopathology because of infection-related inflammation, 3) the toxic effects of parasite excretory/secretory molecules and 4) the disruption of the bile homeostasis. Furthermore, we and others have recently identified a potentially new risk factor for CCA, especially in China, Taiwan, South Korea and Singapore. Through mutation signature analysis on somatic genomic data, we observed an association of CCA with the herbal carcinogen Aristolochic Acid (AA), which is a natural compound exclusively found in plants of the Aristolochia genus used in traditional remedies for numerous indications. With the goal of better understanding the underlying oncogenic mechanisms in CCA, we characterized and compared the genetic and epigenetic changes in OV and non-OV driven and AA-related CCAs from different populations. We found that in non-OV driven CCA, mutations in epigenetic modifier genes including IDH1/2 and BAP1 are the main drivers whereas OV-infected tumors are characterized by frequent p53 mutations. These mutational differences set the stage for more in-depth epigenomic characterization and mechanistic dissection of the tumorigenic process. Our recent data indicate that OV driven CCAs display predominantly CpG Island DNA hypermethylation whereas non-OV driven CCAs are predominantly CpG Shore DNA hypermethylated. OV and non-OV CCAs also differ in their enhancer landscape: OV CCAs exhibit widespread activation of inflammatory response genes, while genes involved in maintenance of cell junction are disrupted in non-OV CCAs. Several lines of evidence also support that chronic inflammation can lead to aberrant DNA methylation and de novo enhancer activity. Consequently, chronic inflammation associated with OV infection may play a key role in setting up divergent enhancer landscapes between OV-related and non-OV CCAs. Other than OV and non-OV driven CCAs, AA represents a profound public health risk in Asia due to its wide use in herbal remedies. We found that AA exposure causes unique mutational signature of A:T to T:A transversion, with an antisense strand bias. We recently detected AA signature in many CCA cases, especially from Asian countries. At present, we are conducting epigenomics studies on AA-related CCAs to understand how AA may impact the epigenomic landscape of CCA. Our multi-omics analysis reflects the contrasting etiologies of CCA, strongly positing that the same disease manifestation can be in fact driven by vastly different molecular circuitries and that each patient should be treated with appropriately targeted therapy. These findings will deepen our understanding of early transformation events in CCA, which may lead to the development of relevant biomarkers and treatment regimens for each subgroup of CCA. As such, our study illustrates the value of investigating specific cancers in the context of diverse populations and etiologies. Citation Format: Bin Tean Teh. Genomic and epigenomic studies of cholangiocarcinoma in diverse populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr PL02-04.

The inconspicuous gatekeeper: endophytic Serendipita vermifera acts as extended plant protection barrier in the rhizosphere.

In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant-microbe communities, the mechanisms driving such multipartite interactions remain largely unknown. Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil-based split-root system. Fungal confrontation in soil resulted in induction of B.sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S.vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S.vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S.vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response. The activation of stress response genes and concomitant repression of putative effector gene expression in B.sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen's virulence potential before host plant infection.

Response of sugar metabolism in apple leaves subjected to short-term drought stress

For a comprehensive understanding of gene expression, enzyme activity and sugar concentrations in response to short-term water deficit in apple (Greensleeves), sugar-modulated gene expression and enzyme activities were analyzed. Water stress resulted in the accumulation of sorbitol, glucose, fructose, galactose and starch, accompanied by a significant reduction in photosynthesis and sucrose concentration. In response to short-term water deficits, the activities of aldose-6-phosphate reductase (A6PR; EC 1.1.1.200), sorbitol dehydrogenase (SDH; EC 1.1.1.14), neutral invertase (NINV; EC 3.2.1.26), sucrose synthase (SUSY; EC 2.4.1.13), and fructokinase (FK; EC 2.7.1.4) were higher, whereas cell wall invertase (CWINV; EC 3.2.1.26) and hexokinase (HK; EC 2.7.1.1) activities were lower. In addition, sucrose phosphate synthase (SPS; EC 2.4.1.14) activity increased during the initial stages of dehydration and then decreased as the drought strengthened. Transcript levels of MdA6PR, MdSDH1/2, MdNINV1/2, MdSUSY3, MdFK1/2/4, MdSOT1/2, MdSUC1-3, MdTMT2/3, MdvGT1, MdpGlcT1-4 were upregulated, whereas transcript levels of MdCWINV1/2, MdHK1/2/3/5, and MdTMT1 were downregulated after 6 days of water stress. These findings suggest that the sorbitol metabolism pathway is induced and high levels of hexose derived from photosynthetic products are transported into vacuoles for adjustment to the water deficit. Our results provide insights into the relationships between sugar levels and sugar-modulated gene and enzyme activity in response to the imposition of short-term water stress.

MON-201 ER Stress Activated by Androgen Enhances Apoptosis of Granulosa Cells via Induction of Death Receptor 5 in PCOS

PCOS is associated with hyperandrogenism and growth arrest of antral follicles. Excess androgens work as a stage-specific inhibitor of a follicular growth suppressing the growth of late-stage follicles. Previously, we found that endoplasmic reticulum (ER) stress is activated in granulosa cells of antral follicles in PCOS (1). This is evidenced by the activation of various unfolded protein response (UPR) genes, including a pro-apoptotic UPR transcription factor, C/EBP homologous protein (CHOP). Death receptor (DR) 5, which harbors a CHOP-binding site in its promoter region, plays a crucial role in ER stress-induced apoptosis by increasing autocrine death-ligand signal, independent of its extracellular ligand TNF-related apoptosis-inducing ligand (TRAIL) (2). Based on these observations, we hypothesized that ER stress is activated by androgen in granulosa cells of antral follicles in PCOS and activated ER stress promotes apoptosis via induction of CHOP and subsequent activation of DR5. Using RT-PCR (qPCR), we showed that testosterone induced the mRNA expression of various UPR factors including XBP1(S), HSPA5, ATF4 and CHOP along with DR5 in cultured human granulosa-lutein cells (GLCs). Treatment with an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), inhibited testosterone-induced mRNA and protein expression of DR5 and CHOP, with a concomitant reduction in apoptosis examined by qPCR, western blotting and flow cytometry respectively. Furthermore, knockdown of CHOP or DR5 by RNA interference inhibited testosterone-induced apoptosis and knockdown of CHOP reduced testosterone-induced DR5 mRNA expression. Treatment with an androgen receptor (AR) antagonist, flutamide, as well as the knockdown of AR, decreased testosterone-induced DR5 and CHOP mRNA expression and apoptosis. In addition, expression of DR5 and CHOP was upregulated in GLCs of PCOS patients and granulosa cells of antral follicles in ovarian sections obtained from both PCOS patients and dehydroepiandrosterone (DHEA)-induced PCOS mice, proven by qPCR and immunohistochemistry (IHC). Treatment of PCOS mice with TUDCA decreased apoptosis and DR5 expression in granulosa cells of antral follicles, with a concomitant reduction in CHOP expression, shown by TUNEL and IHC staining. Our findings indicate that ER stress activated by hyperandrogenism in PCOS promotes apoptosis of granulosa cells of antral follicles via induction of DR5. Reference: (1) Takahashi N, Harada M, et al. Sci Rep 2017, 7:10824 (2) Lu M et al. Science 2014;345:98-101.

SUN-013 Estrogen Receptor Alpha Transactivation Function 1 Dependent Uterotropic Estrogenic Genes

Estrogen receptor alpha (ERα) is a ligand dependent transcription regulator, which contains two transactivation domains, AF-1 and AF-2. The activities of these domains are differentially regulated by various estrogenic signals. The chemicals which control the activities of ERα AF-1 and AF-2 in a discriminatory manner in target tissues are named Selective Estrogen Receptor Modulators (SERMs). However, there is very little information on AF-1 or AF-2 predominantly regulated genes within tissues. Therefore, we sought out to determine which estrogenic genes are regulated by AF-1 using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an estrogen-insensitive mutant ERα but unique to this mutant, AF-1 can be activated by estrogen-antagonists, such as fulvestrant (ICI) and 4-hydroxytamoxifen, in vitro and in vivo. Uterine microarray analysis revealed that the several genes showed higher fold activation level in the ICI-treated AF2ERKI compared to the estradiol (E2)-treated WT. The genes belong to such category were considered as potential AF-1 regulated genes. We analyzed the promotor activity of estrogen responsive uterine genes using an in vitro cell based luciferase reporter assay using the HepG2 cells. In order to define the promoter functionality, we selected Klk1b21, which fold activation level was higher in the ICI-treated AF2ERKI than E2-treated WT uterus, and Mad2l1, which fold activation level was higher in the E2-treated WT than ICI-treated AF2ERKI uterus. We generated the luciferase reporter plasmids which contain 1k bp 5’-flanking sequence of Klk1b21 or the 1k bp 5’-flanking sequence of Mad2l1. The promoter activity of Klk1b21 was enhanced by AF2ER with ICI and WT-ERα with E2 at the same level. On the other hand, Mad2l1 promoter was activated by WT-ERα with E2 but not AF2ER with ICI. These results suggest that Klk1b21 gene is likely to be regulated by AF-1 predominantly. Further analyses revealed that an 80 bp element of Klk1b21 gene, containing a transcription initiator element (Inr) and a TATA-like sequence contributes to the AF-1 preferential activity of the gene, rather than the sequence of the ERα binding element. These results suggest that the involvement of ERα AF-1 and AF-2 varies in the regulation of each estrogen responsive gene. The AF2ERKI mouse is a useful model to define the AF-1 predominantly regulated genes in the estrogen target tissues. The information would enhance the understanding of the mechanism of selective functionality of SERMs in target organs.

MYB96 recruits the HDA15 protein to suppress negative regulators of ABA signaling in Arabidopsis

Unlike activation of target genes in response to abscisic acid (ABA), how MYB96 transcription factor represses ABA-repressible genes to further enhance ABA responses remains unknown. Here, we show MYB96 interacts with the histone modifier HDA15 to suppress negative regulators of early ABA signaling. The MYB96-HDA15 complex co-binds to the promoters of a subset of RHO GTPASE OF PLANTS (ROP) genes, ROP6, ROP10, and ROP11, and represses their expression by removing acetyl groups of histone H3 and H4 from the cognate regions, particularly in the presence of ABA. In support, HDA15-deficient mutants display reduced ABA sensitivity and are susceptible to drought stress with derepression of the ROP genes, as observed in the myb96-1 mutant. Biochemical and genetic analyses show that MYB96 and HDA15 are interdependent in the regulation of ROP suppression. Thus, MYB96 confers maximal ABA sensitivity by regulating both positive and negative regulators of ABA signaling through distinctive molecular mechanisms.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L.).

BackgroundHistone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet.ResultsIn this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf.ConclusionsWe have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

Salicylic acid and nitric oxide signaling in plant heat stress.

In agriculture, heat stress (HS) has become one of the eminent abiotic threats to crop growth, productivity and nutritional security because of the continuous increase in global mean temperature. Studies have annotated that the heat stress response (HSR) in plants is highly conserved, involving complex regulatory networks of various signaling and sensor molecules. In this context, the ubiquitous-signaling molecules salicylic acid (SA) and nitric oxide (NO) have diverted the attention of the plant science community because of their putative roles in plant abiotic and biotic stress tolerance. However, their involvement in the transcriptional regulatory networks in plant HS tolerance is still poorly understood. In this review, we have conceptualized current knowledge concerning how SA and NO sense HS in plants and how they trigger the HSR leading to the activation of transcriptional-signaling cascades. Fundamentals of functional components and signaling networks associated with molecular mechanisms involved in SA/NO-mediated HSR in plants have also been discussed. Increasing evidences have suggested the involvement of epigenetic modifications in the development of a 'stress memory', thereby provoking the role of epigenetic mechanisms in the regulation of plant's innate immunity under HS. Thus, we have also explored the recent advancements regarding the biological mechanisms and the underlying significance of epigenetic regulations involved in the activation of HS responsive genes and transcription factors by providing conceptual frameworks for understanding molecular mechanisms behind the 'transcriptional stress memory' as potential memory tools in the regulation of plant HSR.

Staphylococcal Superantigens: Pyrogenic Toxins Induce Toxic Shock

Staphylococcal enterotoxin B (SEB) and related superantigenic toxins produced by Staphylococcus aureus are potent activators of the immune system. These protein toxins bind to major histocompatibility complex (MHC) class II molecules and specific Vβ regions of T-cell receptors (TCRs), resulting in the activation of both monocytes/macrophages and T lymphocytes. The bridging of TCRs with MHC class II molecules by superantigens triggers an early “cytokine storm” and massive polyclonal T-cell proliferation. Proinflammatory cytokines, tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 elicit fever, inflammation, multiple organ injury, hypotension, and lethal shock. Upon MHC/TCR ligation, superantigens induce signaling pathways, including mitogen-activated protein kinase cascades and cytokine receptor signaling, which results in NFκB activation and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. In addition, gene profiling studies have revealed the essential roles of innate antimicrobial defense genes in the pathogenesis of SEB. The genes expressed in a murine model of SEB-induced shock include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, endoplasmic reticulum/mitochondrial stress responses, immunoproteasome components, and IFN-stimulated genes. This review focuses on the signaling pathways induced by superantigens that lead to the activation of inflammation and damage response genes. The induction of these damage response genes provides evidence that SEB induces danger signals in host cells, resulting in multiorgan injury and toxic shock. Therapeutics targeting both host inflammatory and cell death pathways can potentially mitigate the toxic effects of staphylococcal superantigens.

3448 Macrophages, APOL1 Genotype, & Immunometabolism in CVD (MAGIC

OBJECTIVES/SPECIFIC AIMS: This study aims to understand the potential immunomudualtory effect of APOL1 variants in auto-antigen activated myeloid cells by assessing lysosomal integrity in activated cells expressing APOL1. The primary stimuli were: 1. ssRNA hY3 as a proxy for the Ro immune complex; 2. in an bulk RNA seq model, interferon-response gene, Siglec 1, as a read out of interferon activity. The primary outcomes were: 1. Myeloid cell APOL1 expression both in primary macrophage cultures and ex-vivo patient derived macrophages; 2. Lysosome integrity as measured by fluorescence intensity of lysotracker dye on light microscopy. METHODS/STUDY POPULATION: All recruited subjects provided written informed consent as per the NEW YORK UNIVERSITY Division of Rheumatology-wide Specimen and Matched Phenotype Linked Examination (SAMPLE) protocol. Subjects were African American; SLE subjects met 4 American College of Rheumatology criteria for SLE. Healthy donor monocytes representing each genotype in duplicate (reference allele: G0/G0; heterozygote variant: RV/G0; and homozygote variant RV/RV) were cultured with GM-CSF to yield macrophages which were incubated in serum free media or with hY3 ssRNA (TLR 7/8 agonist) to yield inflammatory M1 macrophages. Fold increase of APOL1 in untreated vs hY3 treated macrophages was measured using qPCR. Live cells were then cultured on glass chamber slides with DNA dye, DAPI, and Lysotracker red, a fluorescent dye that stains acidic lysosomes. As a proof of concept, interferon response gene, Signlec1, and APOL1 transcriptional activity in peripheral blood monocytes (PBMCs) were measured and correlated in 17 SLE patients by RNA seq. RESULTS/ANTICIPATED RESULTS: Regardless of genotype, hY3 increased APOL1 expression by 29 (+/−18.4) fold (P = 0.007 vs no treatment). Genotyping of the qPCR product showed concordance with the chromosomal DNA with the RV heterozygotes expressing both alleles. To examine lysosomal membrane integrity, live hY3-treated macrophages were stained with lysosotracker dye and fluorescence intensity was measured. Compared to reference allele carrying macrophages, each additional variant allele corresponded with a lesser degree of lysosome compartment staining. In SLE PBMCs, we found that APOL1 was highly expressed, and significantly correlated with Siglec1 (F=10.5; P = 0.005) supporting an association between circulating interferons and APOL1 accumulation in monocytes. DISCUSSION/SIGNIFICANCE OF IMPACT: Given that the “cytokine milieu” in SLE elicits APOL1 expression, induces inflammatory cell metabolic rewiring, and stimulates autophagy thereby exposing defects in autophagic flux, this gene-environment interaction may underpin the relationship between chronic inflammation and heightened APOL1 polymorphism-attributed cardiovascular risk. These data support further inquiry into the intersection between chronic autoimmunity and APOL1’s functional role in the vascular microenvironment. The in vitro studies herein extend our prior work by demonstrating a mechanistic link between SLE-associated inflammation, APOL1 risk variant status and CVD via a lysosomal defect which converges on common autophagic and metabolic pathways in mononuclear cells.

Dual-specificity phosphatase 4 is upregulated during skeletal muscle atrophy and modulates extracellular signal-regulated kinase activity.

Skeletal muscle atrophy results from disparate physiological conditions, including denervation, corticosteroid treatment, and aging. The purpose of this study was to describe and characterize the function of dual-specificity phosphatase 4 (Dusp4) in skeletal muscle after it was found to be induced in response to neurogenic atrophy. Quantitative PCR and Western blot analysis revealed that Dusp4 is expressed during myoblast proliferation but rapidly disappears as muscle cells differentiate. The Dusp4 regulatory region was cloned and found to contain a conserved E-box element that negatively regulates Dusp4 reporter gene activity in response to myogenic regulatory factor expression. In addition, the proximal 3'-untranslated region of Dusp4 acts in an inhibitory manner to repress reporter gene activity as muscle cells progress through the differentiation process. To determine potential function, Dusp4 was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize to the nucleus of proliferating myoblasts. Furthermore, Dusp4 overexpression delayed C2C12 muscle cell differentiation and resulted in repression of a MAP kinase signaling pathway reporter gene. Ectopic expression of a Dusp4 dominant negative mutant blocked muscle cell differentiation and attenuated MAP kinase signaling by preferentially targeting the ERK1/2 branch, but not the p38 branch, of the MAP kinase signaling cascade in skeletal muscle cells. The findings presented in this study provide the first description of Dusp4 in skeletal muscle and suggest that Dusp4 may play an important role in the regulation of muscle cell differentiation by regulating MAP kinase signaling.

Gracilaria dura extract confers drought tolerance in wheat by modulating abscisic acid homeostasis

Water stress severely reduces the production of wheat. Application of seaweed extracts have started to show promise in protecting plants from environmental stresses as they contain several biostimulants. However, the modes of action of these biostimulants are not clear. Here, we investigated the role of Gracilaria dura (GD), a red alga, in conferring stress tolerance to wheat during drought under glasshouse and agro-ecological conditions by integrating molecular studies with physiological and field investigations. GD-sap application conferred drought tolerance (as the biomass increased by up to 57% and crop yield by 70%), via facilitating physiological changes associated to maintaining higher water content. GD-sap application significantly increased ABA accumulation (2.34 and 1.46 fold at 4 and 6 days of drought, respectively) due to enhanced expression of biosynthesis genes. This followed an activation of ABA response genes and physiological processes including reduced stomatal opening, thus reducing water loss. Moreover, GD-sap application enhanced the expression of stress-protective genes specifically under water stress. Treatment with fluridone, an ABA inhibitor, further support the role of ABA in GD-sap mediated drought tolerance in wheat. The findings of this study provide insights into the functional role of GD-sap in improving drought tolerance and show the potential to commercialize GD-sap as a potent biostimulant for sustainable agriculture in regions prone to drought.

Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis

Although joint injury itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), and the production of inflammatory cytokines. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Our objective was to determine whether inhibition of cdk9-dependent early response gene activation affects changes in the joint metabolome. To model post-traumatic osteoarthritis, we subjected mice to non-invasive Anterior Cruciate Ligament (ACL)-rupture joint injury. Following injury, mice were treated with flavopiridol - a potent and selective inhibitor of Cdk9 kinase activity - to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1h after injury. We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism, anandamide, and others. Inhibition of primary response gene activation immediately after injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites. Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses. We identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not. These data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury, which may offer novel targets for cell-mediated secondary joint damage.

Target of rapamycin, a master regulator of multiple signalling pathways and a potential candidate gene for crop improvement

The target of rapamycin (TOR) protein regulates growth and development in photosynthetic and non-photosynthetic eukaryotes. Although the TOR regulatory networks are involved in nutrient and energy signalling, and transcriptional and translational control of multiple signalling pathways, the molecular mechanism of TOR regulation of plant abiotic stress responses is still unclear. The TOR-mediated transcriptional regulation of genes encoding ribosomal proteins (RP) is a necessity under stress conditions for balanced growth and productivity in plants. The activation of SnRKs (sucrose non-fermenting-related kinases) and the inactivation of TOR signalling in abiotic stresses is in line with the accumulation of ABA and transcriptional activation of stress responsive genes. Autophagy is induced under abiotic stress conditions, which results in degradation of proteins and the release of amino acids, which might possibly induce phosphorylation of TOR and, hence, its activation. TOR signalling also has a role in regulating ABA biosynthesis for transcriptional regulation of stress-related genes. The switch between activation and inactivation of TOR by its phosphorylation and de-phosphorylation maintains balanced growth in response to stresses. In the present review, we discuss the important signalling pathways that are regulated by TOR and try to assess the relationship between TOR signalling and tolerance to abiotic stresses in plants. The review also discusses possible cross-talk between TOR and RP genes in response to abiotic stresses.

Targeted Disruption of the Cohesin Subunit STAG2 Leads to Loss of Insulation and Inappropriate Gene Activation in Response to Differentiation Signals

Cohesin is a multiprotein complex that mediates sister chromatid cohesion during S phase of the cell cycle, but it also plays crucial roles in genome organization and transcription in interphase. Cohesin's role in regulation of the genome helps determine lineage identity and differentiation of hematopoietic cells (Viny et al., JEM, 2015; Mullenders et al., JEM, 2015; Mazumdar et al., Cell stem cell, 2015; Galeev et al., Cell reports, 2016). Mitotic cohesin is composed of RAD21, SMC1A, SMC3 and STAG1/STAG2 subunits. Mutations in the subunits of cohesin complex are present in significant proportion of myeloid malignancies (Thota et al., Blood 2014). In particular, the frequency of cohesin subunit mutation is as high as ~50% in Down-syndrome associated megakaryoblastic leukemia subtype (Yoshida et al., Nature Genetics, 2013).To further understand cohesin's role in gene regulation in a leukemia context, we used CRISPR-CAS9 to engineer a leukemia associated STAG2 mutation, R614*, into K562 leukemia cells to generate isogenic model lines. Homozygous mutant K562 cells showed complete loss of STAG2 expression. We also obtained STAG2 R614* heterozygous cell clones that showed partial loss of STAG2 expression (STAG2 is on the X chromosome, and the wild type allele is mosiacally silenced in heterozygotes).Phenotype analyses of STAG2 mutant K562 cells showed that homozygous mutants had altered morphology and had acquired adherent properties. Homozygous mutant cells activated expression of the stem cell marker, KIT, and showed a reduction in the neutrophil differentiation marker, CD15.We used ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to examine changes in chromatin accessibility, and RNA sequencing to determine changes in gene expression in STAG2 mutants. More than 5,000 genes were dysregulated in STAG2 homozygous mutants, accompanied by profound changes in chromatin accessibility with gain and loss of accessibility represented equally. Loss of accessibility was observed at insulators that recruit CCCTC-binding factor (CTCF), indicating potential loss of insulation at chromatin boundaries. Conversely, gain of chromatin accessibility was observed at regions that are normally repressed in K562, and this was accompanied by activation of transcription factors such as ERG and FLI1. Gain of accessibility was also observed at myeloid-associated super enhancers of the RUNX1 gene.We hypothesized that loss of STAG2 leads to an inability to constrain transcriptional programs upon differentiation of leukemia cells. To test this hypothesis, we stimulated the STAG2 mutant and parental K562 cells with phorbol myristate acetate (PMA) to induce megakaryocyte differentiation. PMA-stimulated STAG2 homozygous mutant cells showed a striking precocious spike in RUNX1 expression that was associated with enhanced transcription from its proximal P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, the spike in transcription of these genes was not observed at 48 hours, indicating that the ectopic transcription of RUNX1 and ERG is confined to early response to stimulation. The BET inhibitor JQ1, was previously shown to block enhancer activity (Bhagwat et al., Cell reports, 2016). Treatment of STAG2 mutant cells with JQ1 dampened ectopic RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG activation during PMA stimulation. These results suggest that ectopic RUNX1 expression in response to STAG2 deficiency is enhancer-driven. Furthermore, JQ1 treatment caused a 2-fold decrease in KIT levels in STAG2-deficient cells, indicating a reversal of immature properties in these cells.In summary, our results suggest that STAG2 mutation results in loss of chromatin insulation, leading to inappropriate activation of hematopoietic enhancers. Loss of constraints on enhancer-driven transcription could in turn underlie the aberrant gene expression in STAG2-deficient cells exposed to differentiation cues. DisclosuresNo relevant conflicts of interest to declare.