Global Repression Research Articles

Eosinophil-mediated suppression and anti–IL-5 enhancement of plasmacytoid dendritic cell interferon responses in asthma

Virus-induced IFN-α secretion by plasmacytoid dendritic cells (pDCs) is negatively impacted by IgE and has been linked to asthma exacerbations. Eosinophils, another contributor to type 2 inflammation, are also associated with asthma severity. We sought to investigate the impact of eosinophils on pDC antiviral interferon responses and determine whether anti-IL-5/5Rα therapy enhances pDC antiviral function. Blood pDCs purified from anonymous donors were stimulated invitro with rhinovirus (RV)-16 in the presence or absence of eosinophils/eosinophil supernatants. IFN-α was measured in supernatants and RNA collected for bulk RNA-sequencing. Next, purified pDCs from 8 individuals with moderate to severe asthma, treated or not treated with anti-IL-5/5Rα therapy, were cultured exvivo with or without RV; IFN-α secretion and differential gene expression analysis were compared between groups. Exposure to either eosinophils or eosinophil supernatants inhibited RV-induced pDC IFN-α secretion in a dose-dependent manner and did not impact pDC viability. Eosinophil-derived neurotoxin and TGF-β partially recapitulated pDC IFN-α inhibition. Transcriptome analysis revealed global repression of pDC interferon response patterns by eosinophils, most notably in basal expression of interferon-stimulated genes. Increased RV-induced IFN-α secretion and transcription as well as increased basal interferon-stimulated gene expression was detected in pDCs from participants treated with anti-IL-5/5Rα therapy. Our findings highlight a novel mechanism through which type 2 inflammation regulates pDC IFN-α responses relevant to RV respiratory infections in the context of eosinophilic airway disease, suggesting a potential mechanism through which eosinophil-depleting therapies may reduce severity of RV illnesses.

Abstract PD3-04: Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype

Abstract Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology and immune composition. TNBCs display transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Using integrative analyses of transcriptomic, epigenetic, proteomic and phospho-proteomic patterns we have identified subtype-specific vulnerabilities, which advanced our understanding of the cellular origins of TNBC subtypes. In mesenchymal subtype tumors we observed high mutation load and genomic instability, absence of immune cells, low PD-L1 expression, loss of global DNA methylation and transcriptional repression of antigen presentation genes through the polycomb repressor complex 2 (PRC2). Using cell line models, we demonstrate that MHC-I expression is epigenetically silenced by the PRC2 in mesenchymal TNBC cell lines and inhibition of EZH2 restores expression. Pharmacological inhibition of EZH2 enhances chemotherapy efficacy in syngeneic murine tumor models providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors. Citation Format: Brian D Lehmann, Antonio Colaprico, Tiago C Silva, Jianjiao Chen, Hanbing An, Yuguang Ban, Lily Wang, Jamaal L James, Justin Balko, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bing Zhang, Jennifer A Pietenpol, Xi S Chen. Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-04.

Exploiting the tumor-suppressive activity of the androgen receptor by CDK4/6 inhibition in castration-resistant prostate cancer

The androgen receptor (AR) plays a pivotal role in driving prostate cancer (PCa) development. However, when stimulated by high levels of androgens, AR can also function as a tumor suppressor in PCa cells. While the high-dose testosterone (high-T) treatment is currently being tested in clinical trials of castration-resistant prostate cancer (CRPC), there is still a pressing need to fully understand the underlying mechanism and thus develop treatment strategies to exploit this tumor-suppressive activity of AR. In this study, we demonstrate that retinoblastoma (Rb) family proteins play a central role in maintaining the global chromatin binding and transcriptional repression program of AR and that Rb inactivation desensitizes CRPC to the high-dose testosterone treatment invitro and invivo. Using a series of patient-derived xenograft (PDX) CRPC models, we further show that the efficacy of high-T treatment can be fully exploited by a CDK4/6 inhibitor, which strengthens the chromatin binding of the Rb-E2F repressor complex by blocking the hyperphosphorylation of Rb proteins. Overall, our study provides strong mechanistic and preclinical evidence on further developing clinical trials to combine high-T with CDK4/6 inhibitors in treating CRPC.

Mechanism of transcription regulation by Acinetobacter baumannii HpaR in the catabolism of p-hydroxyphenylacetate.

HpaR is a transcription regulator in the MarR family that controls the expression of the gene cluster responsible for conversion of p-hydroxyphenylacetate to pyruvate and succinate for cellular metabolism. Here, we report the biochemical and structural characterization of Acinetobacter baumannii HpaR (AbHpaR) and its complex with cognate DNA. Our study revealed that AbHpaR binds upstream of the divergently transcribed hpaA gene and the meta-cleavage operon, as well as the hpaR gene, thereby repressing their transcription by blocking access of RNA polymerase. Structural analysis of AbHpaR-DNA complex revealed that the DNA binding specificity can be achieved via a combination of both direct and indirect DNA sequence readouts. DNA binding of AbHpaR is weakened by 3,4-dihydroxyphenylacetate (DHPA), which is the substrate of the meta-cleavage reactions; this likely leads to expression of the target genes. Based on our findings, we propose a model for how A. baumannii controls transcription of HPA-metabolizing genes, which highlights the independence of global catabolite repression and could be beneficial for metabolic engineering toward bioremediation applications. DATABASES: The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7EL2/pdb and https://doi.org/10.2210/pdb7EL3/pdb for AbHpaR and AbHpaR-DNA complex, respectively.

Correction for Li et al., "Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis".

Volume 12, issue 5, e01192-21, 2021, https://doi.org/10.1128/mBio.01192-21. In Fig. 2B of the original paper, the image of the Western blot that shows the expression level of c15850 using anti-His monoclonal antibody was duplicated in the panel for c21220. This duplication issue happened at the figure assembly stage and was not identified at submission and proof stages. The authors provide here Fig. 2B with the correct Western blot image in the c21220 panel, which does not change results and conclusions of the article. Nonetheless, the authors apologize to the readers for any inconvenience that this error may have caused.

MED12 and BRD4 cooperate to sustain cancer growth upon loss of mediator kinase

Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.

The Temperature-Dependent Expression of the High-Pathogenicity Island Encoding Piscibactin in Vibrionaceae Results From the Combined Effect of the AraC-Like Transcriptional Activator PbtA and Regulatory Factors From the Recipient Genome.

The high-pathogenicity island irp-HPI is widespread among Vibrionaceae encoding the piscibactin siderophore system. The expression of piscibactin genes in the fish pathogen Vibrio anguillarum is favored by low temperatures. However, information about the regulatory mechanism behind irp-HPI gene expression is scarce. In this work, in-frame deletion mutants of V. anguillarum defective in the putative regulators AraC1 and AraC2, encoded by irp-HPI, and in the global regulators H-NS and ToxRS, were constructed and their effect on irp-HPI gene expression was analyzed at 15 and 25°C. The results proved that only AraC1 (renamed as PbtA) is required for the expression of piscibactin biosynthesis and transport genes. PbtA inactivation led to an inability to grow under iron restriction, a loss of the outer membrane piscibactin transporter FrpA, and a significant decrease in virulence for fish. Inactivation of the global repressor H-NS, which is involved in silencing of horizontally acquired genes, also resulted in a lower transcriptional activity of the frpA promoter. Deletion of toxR-S, however, did not have a relevant effect on the expression of the irp-HPI genes. Therefore, while irp-HPI would not be part of the ToxR regulon, H-NS must exert an indirect effect on piscibactin gene expression. Thus, the temperature-dependent expression of the piscibactin-encoding pathogenicity island described in V. anguillarum is the result of the combined effect of the AraC-like transcriptional activator PbtA, harbored in the island, and other not yet defined regulator(s) encoded by the genome. Furthermore, different expression patterns were detected within different irp-HPI evolutionary lineages, which supports a long-term evolution of the irp-HPI genomic island within Vibrionaceae. The mechanism that modulates piscibactin gene expression could also be involved in global regulation of virulence factors in response to temperature changes.

Inhibition of S-Adenosylmethionine Synthesis Promotes Erythropoiesis Via Epigenetic Modifications

Erythroid differentiation involves global gene expression repression, chromatin condensation and enucleation, mitochondria removal and other marked cellular changes. Given the necessity for these dynamic alterations, it is hardly surprising that epigenetic modifications possess important roles for erythropoiesis. S-adenosylmethionine (SAM), a principle methyl donor for DNA and histone methylations, would be involved in this process. Yet little is known about the specific roles for SAM synthesis in erythropoiesis.SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a and we evaluated the in vivo role of SAM synthesis by treating wild type mice (C57BL/6) with a selective Mat2a inhibitor (cycloleucine). As expected, the Mat2a inhibitor administration (henceforth Mat2ai) reduced SAM in bone marrow (BM) cells (SAM; 3.17±0.43 and 0.93±0.10 area ratio for ctrl and Mat2ai, p < 0.01, n = 4 mice). Interestingly, Mat2ai increased erythropoiesis in BM (Ter119 + cell; 46.3±3.1 and 116.4±14.2×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice) and in blood (hemoglobin concentrations in peripheral blood; 13.7±0.18 and 16.3±0.26 g/dl for ctrl and Mat2ai, p < 0.01, n = 8 mice). However, serum erythropoietin concentration decreased (erythropoietin; 254.2±34.1 and 42.7±5.70 pg/ml for ctrl and Mat2ai, p < 0.01, n = 10 mice). Therefore, Mat2ai promoted erythropoiesis in vivo without increasing erythropoietin. To reveal the point where the erythroid differentiation was affected, immature and mature erythroblast subsets in BM were assessed. Although immature erythroblasts were not changed by Mat2ai (24.1±2.80 and 23.8±3.86×10 6 cells for ctrl and Mat2ai, p = 0.95, n = 8 mice), mature erythroblasts in BM increased following Mat2ai (18.9±2.48 and 81.2±9.73×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice). Therefore, Mat2ai promoted erythroid maturation from immature erythroblast in BM. To reveal the mechanistic insight of this promotion of erythroid maturation by Mat2ai, we performed RNA sequencing of immature erythroblast in BM. This analysis revealed that most genes were down-regulated by Mat2ai (differentially expressed genes by Mat2ai; DOWN 2578 genes, UP 72 genes). In line with this notion, transposase-accessible chromatin sequencing (ATAC-seq) of immature erythroblasts revealed that chromatin accessibility was reduced. While DNA methylation analysis (whole genome bisulfite sequence) of immature erythroblasts revealed slightly reduced global DNA methylation (approximately 2%), there were no clear correlations between changes in promotor (or gene-body) DNA methylation and transcription. This result suggests that DNA methylation changes possess limited roles for the erythroid maturation promoted by Mat2ai. On the other hand, we found that an active histone methylation mark (H3K4me3) was selectively reduced by Mat2ai and that the changes of gene expression and H3K4me3 enrichment (revealed by chromatin immunoprecipitation followed by sequencing) correlated (r = 0.66). Therefore, the loss of H3K4me3, but not the DNA methylation, might contribute to the global gene expression repression for erythroid maturation induced by Mat2ai. Finally, in vitro human erythroid differentiation analysis using CD34 + cord blood cells further revealed that therapeutic and genetic inhibition of SAM synthesis induced erythroid maturation, which was cancelled by extracellular administration of SAM. Therefore, SAM synthesis inhibition is a non-erythropoietin trigger for erythroid maturation and this process occurs in human cells.Collectively, we found that SAM synthesis inhibition promoted erythroid maturation in both mouse and human. Histone methylation alteration induced by SAM synthesis inhibition might contribute to this phenomenon. These findings may pave the way to develop a new therapeutic strategy for anemia in erythropoietin independent manner. DisclosuresHarigae: Kyowakirin: Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Ono pharma: Honoraria, Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editas Medicines: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: Sponsored research; FOG Pharma:: Consultancy; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Evidence for reciprocal evolution of the global repressor Mlc and its cognate phosphotransferase system sugar transporter.

Because the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in the regulation of various physiological processes in addition to carbohydrate transport, its expression is precisely regulated in response to the availability of PTS sugars. The PTS consists of enzyme I and histidine phosphocarrier protein, and several sugar-specific enzymes II. In Escherichia coli, genes for enzymes II specific for glucose and related sugars are co-regulated by the global repressor Mlc, and glucose induction of the Mlc regulon genes is achieved by its interaction with glucose-specific enzyme II (EIIGlc ). In this study, we revealed that, in Vibrio species, which are phylogenetically older than Enterobacteriaceae, the membrane sequestration of Mlc and thereby the induction of its regulon genes is mediated by N-acetylglucosamine (NAG)-specific EII. While Vibrio Mlc interacts only with the EIIB domain of EIINag , E. coli Mlc interacts with the EIIB domain of both EIIGlc and EIINag . The present data suggest that EIINag may be the primordial regulator of Mlc, and EIIGlc has evolved to interact with Mlc since an EIIA domain was fused to EIINag in Enterobacteriaceae. Our findings provide insight into the coevolutionary dynamics between a transcription factor and its cognate regulator according to long-term resource availability in the bacterial natural habitat.

Long-Distance Effects of H-NS Binding in the Control of hilD Expression in the Salmonella SPI1 Locus.

Salmonella enterica serovar Typhimurium utilizes a type three secretion system (T3SS) carried on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. HilA activates expression of the T3SS structural genes. Expression of hyper invasion locus A (hilA) is controlled by the transcription factors HilD, HilC, and RtsA, which act in a complex feed-forward regulatory loop. The nucleoid-associated protein H-NS is a xenogeneic silencer that has a major effect on SPI1 expression. In this work, we use genetic techniques to show that disruptions of the chromosomal region surrounding hilD have a cis effect on H-NS-mediated repression of the hilD promoter; this effect occurs asymmetrically over ∼4 kb spanning the prgH-hilD intergenic region. CAT cassettes inserted at various positions in this region are also silenced in relation to the proximity to the hilD promoter. We identify a putative H-NS nucleation site, and its mutation results in derepression of the locus. Furthermore, we genetically show that HilD abrogates H-NS-mediated silencing to activate the hilD promoter. In contrast, H-NS-mediated repression of the hilA promoter, downstream of hilD, is through its control of HilD, which directly activates hilA transcription. Likewise, activation of the prgH promoter, although in a region silenced by H-NS, is strictly dependent on HilA. In summary, we propose a model in which H-NS nucleates within the hilD promoter region to polymerize and exert its repressive effect. Thus, H-NS-mediated repression of SPI1 is primarily through the control of hilD expression, with HilD capable of overcoming H-NS to autoactivate. IMPORTANCE Members of the foodborne pathogen Salmonella rely on a type III secretion system to invade intestinal epithelial cells and initiate infection. This system was acquired through horizontal gene transfer, essentially creating the Salmonella genus. Expression of this critical virulence factor is controlled by a complex regulatory network. The nucleoid protein H-NS is a global repressor of horizontally acquired genomic loci. Here, we identify the critical site of H-NS regulation in this system and show that alterations to the DNA over a surprisingly large region affect this regulation, providing important information regarding the mechanism of H-NS action.

Histone-like nucleoid-structuring protein (H-NS) regulatory role in antibiotic resistance in Acinetobacter baumannii

In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes involved in pathogenesis and stress response. In addition, H-NS inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with β-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns

Chemical splicing modulators that bind to the spliceosome have provided an attractive avenue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. However, the biological effect of proteins containing translated intron sequences remains unclear. Here, we identify a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) via genome-wide ribosome profiling and bio-orthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through c-Jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

Repression of the Orphan Nuclear Receptor Esrrb Results in Glucocorticoid Resistance in ALL

▪Acute lymphoblastic leukemia (ALL) is an aggressive hematological malignancy, in which 20-30% of pediatric patients relapse or undergo induction failure (Vrooman, 2009). ALL is treated with a combination of chemotherapeutic agents, including glucocorticoids (GCs). Pediatric ALL patients who exhibit resistance to GCs at diagnosis or relapse have a poor prognosis(Dördelmann, 1999). We performed an unbiased whole genome survival-based shRNA screen in primary mouse T-ALL cells to identify GC resistance genes. This shRNA screen identified known mediators of GC resistance as well as novel genes involved in chromatin remodeling, cell differentiation and metabolism.One of the top hits in the GC resistance screen were shRNAs targeting the orphan nuclear receptor ESRRB. ESRRB is essential for the maintenance of pluripotency in mouse embryonic stem cells and decreases in ESRRB expression stimulate differentiation (Zhang, 2008). We observe no effects of ESRRB deficiency on leukemic cell growth or differentiation. However, silencing of Esrrb prevents GC-induced apoptosis in mouse and human T-ALL cell lines in vitro . Although the screen was done in mouse T-ALL cells, we find that ESRRB-deficiency also results in GC resistance in human B-ALL cells. As many published GC resistance genes fail to translate in vivo, we transplanted mice with T-ALL cells transduced with shRNAs targeting Esrrb or empty vector. Dexamethasone treatment prevented disease progression in mice transplanted with control T-ALL cells however, dexamethasone treatment had no significant effect on disease development in mice transplanted with ESRRB deficient T-ALL cells, indicating that ESRRB deficiency confers GC resistance in vivo.As ESRRB has no established role in T-cells or the GC response, we performed RNA-sequencing on normal and ESRRB-deficient T-ALL cells in the absence and presence of GCs to uncover the mechanism of GC resistance. ESRRB deficient T-ALL cells fail to upregulate 65% of GC-induced genes, indicating that decreases in ESRRB expression prevents optimal GC-induced gene expression and apoptosis. Furthermore, we identified Esrrb as a GC-induced gene, supporting its role in the GC response. Due to the global repression of GC-induced gene expression in ESRRB-deficient leukemic cells, we examined GC receptor (GR) localization, but found that ESRRB had no detectable effects on GR localization.Collectively these data suggest that ESRRB may function as a novel co-activator of GR mediated transcription. Consistently, we found ESRRB expression decreased in a subset of relapse ALL patients, suggesting that ESRRB may contribute to GC resistance in patients. These findings identify ESRRB as a novel GR co-activator, whose expression is suppressed in a subset of relapse ALL patients. We demonstrate that treatment with the synthetic ESRRB agonist GSK4716 stimulates GC-induced apoptosis and gene expression in human ALL cell lines. These data raise the intriguing possibility that ESRRB agonists may re-sensitize GC resistant leukemia patients to GC therapy. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional changes through menstrual cycle reveal a global transcriptional derepression underlying the molecular mechanism involved in the window of implantation.

The human endometrium is a dynamic tissue that only is receptive to host the embryo during a brief time in the middle secretory phase, called the window of implantation (WOI). Despite its importance, regulation of the menstrual cycle remains incompletely understood. The aim of this study was to characterize the gene cooperation and regulation of menstrual cycle progression, to dissect the molecular complexity underlying acquisition of endometrial receptivity for a successful pregnancy, and to provide the scientific community with detailed gene co-expression information throughout the menstrual cycle on a user-friendly web-tool database. A retrospective gene co-expression analysis was performed based on the endometrial receptivity array (ERarray) gene signature from 523 human endometrial samples collected across the menstrual cycle, including during the WOI. Gene co-expression analysis revealed the WOI as having the significantly smallest proportion of negative correlations for transcriptional profiles associated with successful pregnancies compared to other cycle stages, pointing to a global transcriptional derepression being involved in acquisition of endometrial receptivity. Regulation was greatest during the transition between proliferative and secretory endometrial phases. Further, we prioritized nuclear hormone receptors as major regulators of this derepression and proved that some genes and transcription factors involved in this process were dysregulated in patients with recurrent implantation failure. We also compiled the wealth of gene co-expression data to stimulate hypothesis-driven single-molecule endometrial studies in a user-friendly database: Menstrual Cycle Gene Co-expression Network (www.menstrualcyclegcn.com). This study revealed a global transcriptional repression across the menstrual cycle, which relaxes when the WOI opens for transcriptional profiles associated with successful pregnancies. These findings suggest that a global transcriptional derepression is needed for embryo implantation and early development.

Structure of Gcn1 bound to stalled and colliding 80S ribosomes

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes.

Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender‐specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2‐O3 operates as a transcription repressor in the female gametocytes. AP2‐O3 is specifically expressed in the female gametocytes. AP2‐O3‐deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post‐fertilization. AP2‐O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2‐O3 targets a substantial proportion of the male genes by recognizing an 8‐base DNA motif. In addition, the maternal AP2‐O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female‐specific transcriptome and essential for the mosquito transmission of Plasmodium.

Differential functions of TLE1 and TLE3 depending on a specific phosphorylation site

Mammalian Transducin-like enhancer of split (TLE) confer global repression of numerous target genes in conjunction with a myriad of DNA-binding repressors. These factors have a major role in the regulation of multiple signal transduction pathways. Evidence have been obtained regarding the possible role of some of these proteins in cancer. TLE3 was suggested as a marker for increased chemosensitivity from pathological studies. Here we demonstrate, using the TCGA data base, differences in expression of this gene compared to TLE1 in several cancers. In-vitro transduction of a retrovirus encoding TLE3 to A549 lung cancer cells increased paclitaxel effectivity while TLE1 introduction to these cells decreased it. While TLE1 and TLE3 share ∼80% amino acid identity, we show that mutating or reconstituting an amino-terminal phosphorylation site, which is present only in TLE1 but absent from TLE3, and is evolutionary conserved, converts the activity of TLE1 to that of TLE3 like and vice versa. We repeated these results in an adipocytes differentiation system. Our results reveal how a single phosphorylation site can confer distinct qualitative or quantitative activities on highly homologous transcriptional regulators.

The budding yeast transition to quiescence.

A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence-specific repressors. The final division that takes place post-DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin-dependent, long-range chromatin interactions lead to genome-wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast.

Acute stress drives global repression through two independent RNA polymerase II stalling events in Saccharomyces

SUMMARYIn multicellular eukaryotes, RNA polymerase (Pol) II pauses transcription ~30–50 bp after initiation. While the budding yeast Saccharomyces has its transcription mechanisms mostly conserved with other eukaryotes, it appears to lack this fundamental promoter-proximal pausing. However, we now report that nearly all yeast genes, including constitutive and inducible genes, manifest two distinct transcriptional stall sites that are brought on by acute environmental signaling (e.g., peroxide stress). Pol II first stalls at the pre-initiation stage before promoter clearance, but after DNA melting and factor acquisition, and may involve inhibited dephosphorylation. The second stall occurs at the +2 nucleosome. It acquires most, but not all, elongation factor interactions. Its regulation may include Bur1/Spt4/5. Our results suggest that a double Pol II stall is a mechanism to downregulate essentially all genes in concert.

1192. H-NS-like Proteins in Pseudomonas aeruginosa Coordinately Silence Intragenic and Antisense Transcription

BackgroundThe H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome, which include horizontally acquired genes and numerous virulence factors. Although cells can tolerate the loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of the prophage Pf4 and subsequent superinfection of the cell. Although in other bacteria, H-NS promotes cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase, a role for MvaT and MvaU in repressing transcription from intragenic promoters has not been demonstrated.MethodsHere we utilize a parental strain that cannot be infected by Pf4 phage to identify the combined MvaT and MvaU regulon. RNA-seq was utilized to identify genes differentially expressed in cells lacking MvaU or both MvaU and MvaT. ChIP-seq was utilized to identify genes directly regulated by MvaT and MvaU in wild-type cells. Further, ChIP-seq was performed in cells of the parental strain and cells lacking both MvaT and MvaU to map genome-wide σ 70-dependent promoters that were active in the presence or absence of both H-NS-like proteins.ResultsWe demonstrate that the loss of both MvaT and MvaU, but not MvaU alone, leads to increased sense, antisense, and intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ 70 to those genomic regions vacated by these proteins.Loss of MvaT and MvaU causes global changes in gene expression Loss of MvaT and MvaU results in increased sense and antisense transcription Loss of both MvaT and MvaU results in genome-wide redistribution of RNA polymerase ConclusionOur findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and describe a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.Disclosures All Authors: No reported disclosures