Tspo induced by inflammation is not clear. Here, we revealed that lipopolysaccharide (LPS) -induced Tspo expression was activated by the AP-1 complex in a mouse microglial cell line, BV-2. Knockdown of c-Fos and c-Jun, the components of AP-1, reduced LPS-induced Tspo expression. Furthermore, the enrichment of Sp1 in the proximal promoter region of Tspo was increased in the presence of LPS. In addition, the binding of histone deacetylase 1 (HDAC1) to the enhancer region, which contains the AP-1 site, was decreased by LPS treatment, but there were no significant differences in HDAC1 binding to the proximal promoter region with or without LPS. These results indicated that HDAC1 is involved not in the proximal promoter region but in the enhancer region. Our study revealed that inflammatory signals induce the recruitment of AP-1 to the enhancer region and Sp1 to the proximal promoter region of the Tspo gene and that Sp1 may regulate the basal expression of Tspo.It has been reported that neuroinflammation occurs in the central nervous system (CNS) in patients with neuropathic pain, Alzheimer’s disease and autism spectrum disorder. The 18-kDa translocator protein TSPO is used as an imaging target in positron emission tomography to detect neuroinflammation, and its expression is correlated with microglial activation. However, the mechanism underlying the transcriptional regulation of  Microglia are resident immune cells of the central nervous system (CNS) and play a pivotal role in maintaining the neuronal environment. Resting (ramified) microglia monitor neurons by “palpating” synapses . Individ11C] (R) PK-11195 or [11C] PBR28, is widely used in animal models or human subjects  and plays different roles, for example, in the regulation of cell growth ). siRNAs (10 nM) were transfected into BV-2 cells using Lipofectamine RNAiMAX  in accordance with the manufacturer’s instructions.The c-Fos and c-Jun knockdown experiments were performed using siRNA purchased from Thermo Fisher Scientific .All numerical data are presented as the means ± S. Ds, with n = 3  or n = 4 (reporter gene assay). Comparisons were assessed using Student’s t-test or ANOVA. A value of Tspo mRNA was induced by a high concentration of LPS (100 or 500 ng/ml), peaked at 8 hours and gradually returned to normal. By contrast, the expression of Tspo mRNA did not increase at low concentrations of LPS (1 or 10 ng/ml). Under the same conditions, the expression of pro-inflammatory mediators such as Tnf-α, Il-1β and Il-6 was also upregulated by LPS stimulation . Interestingly, TSPO was also localized in the membrane fraction in the control group and was 1.94-fold higher in the LPS-treated group than in the control group (p = 0.005). To verify whether TSPO is localized in the plasma membrane, TSPO, the Na+/K+ ATPase and mitochondria were stained by immunocytochemistry and analyzed by laser confocal microscopy. As previously reported, almost all TSPO protein was localized in the mitochondrial outer membrane. However, some fluorescence signals corresponding to TSPO protein were detected in non-mitochondrial regions in both the absence and presence of LPS  and prediction tools for transcription binding sites, such as STAMP (http://www.benoslab.pitt.edu/stamp/), JASPAR (http://jaspardev.genereg.net/) and DBCLS Galaxy . The presumed transcription factors and their binding sites were identified via a comprehensive assessment using several tools and are shown in cis element regulates Tspo expression during LPS treatment, serial deletion constructs were designed for the Tspo reporter gene assay according to the schematic in Tspo during LPS stimulation.Prior to the construction of vectors for the reporter gene assay, the Tspo regulatory region was promoted by LPS treatment. A previous study showed that TSPO expression is regulated by the binding of Sp1, Sp3 and/or Sp4 to the proximal promoter region in human breast cancer cell lines, and a similar sequence was observed in the mouse Tspo gene  (R) PK-11195 to TSPO is increased in multiple brain regions, such as the cerebellum, brainstem and anterior cingulate cortex (ACC) Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: N/A**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript could be improved by considering the following points1. I think it would be important to include something about PU.1 in the discussion (The binding motif of PU.1 is in enhancer region (-593-520) and lies 3 bp apart from the AP1 core binding motif). The expression pattern of TSPO in microglia is very specific, and therefore Lineage dependent transcription factors (LDTFs) such as PU.1 have to play a role . Infact, the initial steps of enhancer selection in closed chromatin regions containing regularly positioned nucleosomes involves the binding of LDTFs. This binding results in the depletion/sliding of nucleosomes to expose enhancer DNA sequences. Indeed, in microglia/macrophages, enhancers controlling endotoxin-stimulated gene expression are almost invariably bound by the lineage dependent Ets transcription factor Pu.1 . These LDTFs bound to enhancers induce histone modifications (such as H3K4me1) associated with a primed state of activity . Histone mark H3K4me1 is a core chromatin signature of primed enhancers and serves as a beacon for signal-dependent effectors of signaling pathways such as nuclear factor-κB (NFκB), interferon responsive factors (IRFs) and activator protein 1 (AP-1) .Ghisletti, S., Barozzi, I., Mietton, F., Polletti, S., De Santa, F., Venturini, E., Gregory, L., Lonie, L., Chew, A., Wei, C.-L., et al. (2010). Identification and Characterization of Enhancers Controlling the Inflammatory Gene Expression Program in Macrophages. Immunity 32, 317–328.Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X., Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589.Holtman, I.R., Skola, D., and Glass, C.K. (2017). Transcriptional control of microglia phenotypes in health and disease. J. Clin. Invest. 127, 3220–3229.Smale, S.T., and Natoli, G. (2014). Transcriptional control of inflammatory responses. Cold Spring Harb. Perspect. Biol. 6, a016261.2. It would be important to comment on differences observed in TSPO expression following knockdown of cFos and cJun. In a recent paper :1119-1133.), decreased TSPO expression was only observed following a combined knockdown of cJun and cFos. In the current paper, the authors report a decrease in TSPO expression when either cJun or cFos is knockdown. Is this due to cell-line differences? (I noticed they used a different BV-2 cell-line which was maintained in DMEM medium).3. In paragraph 4 of the discussion, Sp1 transcription factor is discussed. There is no mention of either Sp3 or Sp4, despite Rashid et al. showing that these factors bind the GC-boxes in the proximal promoter  and that knockdown of either Sp3 or Sp4 significantly reduces TSPO promoter activity in BV-2 microglia.4. In paragraph 5 of the discussion, the authors state “ human TSPO expression increases during the embryo to prenatal period and decreases during the neonatal period, and the TSPO expression level in the neonatal period is maintained throughout life. These data suggest that the TSPO functions, such as increasing cholesterol transport from cytosol to mitochondria and promoting pregnenolone production, might be important for fetal development.” While this statement might be true, I think it also important to acknowledge that the increase in TSPO expression during development might simply be a reflection of an activated microglia state. During development, microglia are predominantly in the amoeboid state as they play a major role in removing excessive produced apoptotic neurons and large amounts of extracellular debris from the developing CNS.Reviewer #2: The authors adequately addressed the issues from the original review. The paper describes a data-supported mechanism for transcriptional regulation of TSPO in LPS-treated microglia. A few minor points:1) Regarding M1/M2 classification - it should be noted that the range of microglial activation phenotypes is complex and that the M1/M2 classification in the paper is being used for the sake of simplicity.2) The antibodies and oligonucleotide sequences used can be placed in supplemental information.3) There were a few grammatical errors throughout the manuscript.Reviewer #3: In this manuscript the authors described a potential mechanism of the increased TSPO expression after LPS treatment which were regulated by AP-1 complex and HDAC1 in BV-2 microglia cell at the transcriptional level. I have some specific issues that need clarification.1. The title should be replaced as “Lipopolysaccharide induces mouse translocator protein (18 kDa) expression by AP-1 complex in microglial cell line, BV-2”.2. As the authors stated in the manuscript, the concentration of HDAC1  was decreased in the enhancer region of TSPO at the AP-1 binding site after LPS treatment. I have concerns about the potential effects of HDAC1 intervention on TSPO expression or LPS induced inflammatory reaction.3. Writing of the paper might be improved to meet sufficient quality.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  18 Aug 2019Reviewer 11. Thank you for the constructive comments to improve our study. We should have mentioned about Pu.1/Ets.2 in the discussion. We added the sentences as below . “... transcriptional regulation of Tspo. In addition to the involvement of AP-1 and Sp1, Rashid et al reported that the Pu.1 and its binding motif, Ets, play an important role in the transactivation of Tspo gene expression. Prediction tools for transcription binding sites were used in this study; however, Pu.1/Ets was not identified as a candidate transcription factor for the regulation of the Tspo gene. Contrary to the prediction results, the Ets sequence (5’-GGAA-3’) is located 3 bp from the AP-1 binding site and acts as a transcriptional activator of Tspo in response to LPS stimulation.”2. Thank you for the constructive comments to improve our study. We also wondered the differences between our results and a recent study . Batarseh et al reported that PMA-induced Tspo expression was reduced by the solely knockdown of c-Jun in MA-10 cells and NIH-3T3 cells , hence, it is thought to be that the expression of Tspo was inhibited by the knockdown of either of c-Fos or c-Jun. In case of our study, the protocols such as cell and its culture condition, the dose or time of LPS stimulation and the measurement of gene expression by qRT-PCR or Western blotting were diffed from previous studies, the results were not completely consistent with those studies.3. Thank you for your comments. Rashid et al (Ref. #34) and Batarseh et al (Ref. #26) reported that the enrichment of Sp1, Sp3 and Sp4 were increased at the GC-boxes in the proximal and distal promoter region of Tspo gene and the knockdown experiments of Sp3 or Sp4 significantly reduced the transactivation of Tspo, hence, it is considered to be a certain phenomenon that the involvement of Sp family in the Tspo gene expression.Previous studies showed that Sp1 and HDAC1 forms a transcriptional repressor complex at the transcriptional inactive state. Upon the stimulation, the enrichment of Sp1 did not affect, however, HDAC1 was released from Sp1 and replaced to CBP/p300 (Ref. #21 and 27).We hypothesized that the same molecular mechanism was occurred in the transcriptional regulation of Tspo gene. Furthermore, since there were few studies that the interaction between HDAC1 and Sp3 or Sp4, we excluded Sp3 and Sp4 from this study. The additional information is shown in the results section as described below. “... a, transcriptionally repressed state . Furthermore, since few studies have examined the interaction between HDAC1 and Sp3 or Sp4, we excluded Sp3 and Sp4 from this study and focused on the interaction between Sp1 and HDAC1 in the proximal promoter region of the Tspo gene.”4. Thank you for the constructive comments to improve our study. As you pointed out, we should have mentioned about the activation of microglia during CNS development. We added the sentences in the discussion section with reference :1387-94. Review. PubMed PMID: 17965659) as below.  “... for fetal development. In addition, there is a possibility that the increase in TSPO expression during the neural developmental stage might be reflective of activated microglia. During neural development, microglia are activated and maintain CNS homeostasis by removing apoptotic cells, myelin debris and other endogenous redundant debris [40].”Reviewer 21. Thank you for your advice. As you pointed out, the classification of microglia is complicated according to various studies. We described only classical classification of microglia in introduction section and did not mention detailed classification such as M (LPS) and M (IL-4 and IL-13) (previously named as M2a) that reported recently :1138-59). The objective of this study is clarification of transcriptional mechanism of Tspo gene in mice, hence, we did not intend to mention the classification of microglial cell. LPS and IFN-γ were used for induction of activated microglia and Tspo gene expression as pro-inflammatory inducers.2. Thank you for your advice. The lists of oligonucleotide sequences and antibodies were transferred to S1 and S2 Table.3. Thank you for your advice. We revised the manuscript and was proofread by the Springer Nature Author Services.Reviewer 31. Thank you for your advice. The title was replaced as follows. “Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2.”2. Thank you for your comments. Upon LPS or pro-inflammatory stimulation, various pathways were activated and HDAC1 was released from AP-1 binding site, transcriptional activators were then recruited to the Tspo promoter. Of course, since it is thought that not all HDAC1 molecules were released from AP-1 binding site, some HDAC1 molecules remained at the AP-1 binding site. Furthermore, the enrichment of HDAC1 might gradually returns to normal condition in time-dependent manner after once upon release from AP-1 binding site to inhibit activated gene expression.3. Thank you for your advice. We revised the manuscript and was proofread by the Springer Nature Author Services.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 10 Sep 2019[EXSCINDED]Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2PONE-D-19-15932R1Dear Dr. Nakamura,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have adequately addressed my comments. The language has been improved and the manuscript is now better readable.Reviewer #2: (No Response)**********what does this mean?). 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Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle–independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the  The longer a cell remains in G0, the more refractory it becomes to re-entering the cell cycle. This study shows that in terminally differentiated cells in vivo, regulatory elements at genes encoding just three key cell cycle regulators  become inaccessible, limiting their aberrant activation and maintaining a prolonged, robust G0. The majority of cells in mature multicellular organisms spend most of their existence in nonproliferating states, often referred to as cellular quiescence or the G0 phase . SubstanDrosophila) bind with the dimerization partner (DP) at promoter proximal E2F binding motifs at hundreds of cell cycle genes, including cyclins, cyclin-dependent kinases (Cdks), replication proteins, and mitotic regulators, to promote their expression. Upon entry into G0, silencing through these same binding sites occurs via the formation of a transcriptional repressor complex called DP, retinoblastoma (RB)-like, E2F and MuvB (DREAM). DREAM complexes are conserved from Caenorhabditis elegans to humans, and in Drosophila, DREAM (termed dREAM) consists of the E2F binding partner DP, Retinoblastoma-family protein Rbf1 or Rbf2, the repressive E2F transcription factor family member dE2F2, chromatin assembly factor 1, p55 subunit (p55/CAF1), Myb oncogene-like protein (Myb), and Myb-interacting proteins [The transition from proliferation to G0 is accompanied by a functional switch in the master regulators of the cell cycle program, such as E2F transcription factor (E2F) complexes, leading to global down-regulation of cell cycle gene transcription –6. In prproteins ,6. Formaproteins –10.Although dREAM plays an important role in the repression of cell cycle genes in G0, key aspects of cell cycle exit in vivo are still not understood. For example, in some contexts of differentiation, cells eventually arrest and differentiate even in the presence of constitutive high E2F or cyclin/Cdk activity –15. We aDrosophila wing and found cell cycle exit occurs despite disruption of heterochromatin-dependent gene silencing [Drosophila uses additional mechanisms to ensure a robust G0.We and others have also observed that the longer a terminally differentiated cell remains in G0, the more refractory it becomes to reentering the cell cycle, even in the presence of high E2F or cyclin/Cdk activity . This hailencing  and withDrosophila wing occurs during metamorphosis and is tied to pulses of the hormone ecdysone that induce downstream transcription factors to modulate cell cycle gene expression [Drosophila wing during metamorphosis through RNA sequencing (RNA-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing (FAIRE-seq) over six developmental time points. We show that during wing differentiation, chromatin accessibility and gene expression changes are coordinated with the transition from a proliferating to a postmitotic state. This includes the closing of specific regulatory elements at a subset of critical “master” cell cycle regulators during G0. Moreover, we have uncoupled differentiation from cell cycle exit, revealing that the closing of pupa wing enhancers at these cell cycle master regulator genes is developmentally programmed and occurs independent of E2F activity or cell cycling status, coincident with robust G0. We propose that the developmentally programmed closing of regulatory elements at a subset of key cell cycle genes is the molecular mechanism underlying robust cell cycle exit in vivo.Cell cycle exit in the pression . We recepression . This suDrosophila wings from the late wandering third instar stage when wing cells are proliferating to 44 h after puparium formation (APF), when wing cells are postmitotic and begin to deposit adult cuticle.During metamorphosis, wings undergo morphogenetic changes coordinated with cell cycle alterations, loss of regeneration capacity, and activation of a wing terminal differentiation program –30. ThesWe identified a total of 20,329 high-confidence open chromatin regions (peaks). We first compared the similarity of open chromatin profiles across our wing developmental time course by examining Pearson correlation coefficients. The open chromatin landscape is gradually changing during metamorphosis, and early proliferative stages are clearly distinct from postmitotic stages in chromatin accessibility . By calcConsistent with previous work, our parallel RNA-seq revealed dynamic expression changes for over 6,000 genes (over 35% of the genome) during wing metamorphosis. For comparison to the FAIRE-seq clusters, we also clustered genes based on RNA expression into 18 k-means RNA-seq clusters . ClusterTo more easily visualize the temporal dynamics of peaks, we next compared dynamic peaks between adjacent stages to define them as opening or closing compared with the previous stage , S2 Fig.Drosophila, we find most developmentally dynamic putative regulatory elements for the wing are located within introns, especially the first intron and 1–5 kb upstream of the TSS. This is also consistent with the locations of Drosophila enhancers identified using a functional accessibility-independent approach, self-transcribing active regulatory region sequencing (Starr-Seq) [To correlate chromatin accessibility changes with gene expression changes, we assigned dynamic and static FAIRE peaks to the nearest transcription start site (TSS) . Dynamicarr-Seq) .Open chromatin sites often correspond to gene regulatory elements such as transcriptional enhancers, which can activate or repress gene expression. To determine whether FAIRE peak dynamics correlate positively or negatively with gene expression changes, we assigned each FAIRE peak to its nearest gene and carried out pairwise comparisons between each stage and its next two sequential stages for >2-fold changes in chromatin accessibility correlated with >2-fold expression changes using our RNA-seq data . When weWe observed that the majority of dynamic FAIRE peaks fall into the category of peaks opening with the corresponding gene expression increasing. This suggests that the majority of gene expression changes in the differentiating wing are driven by transcriptional activators gaining access to their binding sites. The second-largest category of FAIRE peaks close and are associated with loss of expression. This suggests that loss of access to transcriptional activators also plays a major role in gene repression during terminal differentiation.We next examined the correlation between gene expression and chromatin accessibility changes during our time course  Figs. WeCuticular protein 51A (Cpr51A) driving upstream activation sequence (UAS)–green fluorescent protein (GFP)  . This reCells in pupal wings exit the cell cycle at 24 h, which accompanies temporally synchronized repression of cell cycle genes. We examined the expression of approximately 300 cell cycle genes compiled from our previous analysis of cell cycle exit  Fig 2E)Fig 2E) aWe carried out a de novo motif discovery on the promoter proximal regions for cell cycle genes using Multiple Em for Motif Elicitation (MEME) tool . The mosDrosophila activator E2F complex E2F1/DP  or E2F + CyclinD (CycD)/Cdk4 to phosphorylate and inactivate Rbf specifically after cells have already established a flexible G0 state at 26 h -GFP transcriptional reporter that includes known E2F binding sites contained within FAIRE peaks that remain accessible after cell cycle exit . At 44 h at 26 h . ExpresscycE, string (stg), and e2f1 fall into this group  or normal GFP (cycE) to capture gene expression shutoff. Our GFP reporters showed dynamic expression correlated with the accessibility of the elements, which verifies some of these distal elements as pupal wing enhancers for these cell cycle genes. However, none of these enhancers individually recapitulate the normal, broad expression of these genes in the pupa wing. Our results suggest that dynamic chromatin accessibility at specific enhancers of complex cell cycle genes drives temporal expression changes during metamorphosis.In contrast to the majority of cell cycle genes, a few key, rate-limiting cell cycle genes are controlled by long, complex regulatory elements upstream of their TSS or in long introns. For example, is group . We findTS) to limit the perturbation of the cell cycle from 12 to 24 or 44 h APF. This allows metamorphosis to initiate properly yet effectively delays G0 by one extra cell cycle or bypasses G0 with multiple rounds of extra division  and complex cell cycle genes (cycE and stg) showed minor changes in chromatin accessibility when cell cycle exit was delayed or disrupted. Importantly, the closing of cycE and stg distal enhancers proceeds with normal timing despite the delay or bypass of cell cycle exit  are also altered in the E2F-only condition, but to a lesser degree than in the E2F + CycD condition. For these genes, we also see accessibility changes when comparing E2F + CycD to the wild-type control, but not when we compare E2F + CycD with E2F only . This isSimilarly, an additional 119 genes in this group are bona fide RB/E2F or dREAM/ Myb-Muv B (MMB) target genes that are only mildly affected in the E2F-only condition and do not pass our RNA-seq cutoff ,56. We fp < 9.81−6) and noncoding RNA (ncRNA) processing (p < 4.76−5), and most of these genes (199) are down-regulated. We do not clearly understand how these genes are regulated by the cell cycle at this time. We speculate that, for these genes, adding CycD/Cdk4 activity could have direct or indirect effects on additional transcriptional regulators that do not alter chromatin accessibility, possibly by acting through or near promoter proximal sites, or CycD/Cdk4 activity could have effects on transcript stability. Consistent with these ideas, this group of genes includes 35 transcription or RNA polymerase II binding factors  and 17 regulators of RNA splicing and stability . We also cannot rule out the possibility that significant changes in RNA-seq signal may result from a small subset of cells in the wing that change gene expression and accessibility but that cannot be captured by the limited dynamic range of the chromatin accessibility signal from the whole tissue.The remaining 354 genes affected specifically when cell cycle exit is bypassed are enriched for GO terms involved in RNA metabolism   . Pupal wpy (TEM) . Chitin Blimp-1, Hormone receptor 3 (Hr3), and crooked legs (crol) were expressed at significantly higher levels at 44 h when cell cycle exit was disrupted, whereas the expression of E74EF, E75B, and E71CD was reduced , Cpr50Ca, Cpr47Ec, and TweedleT (TwdlT) harbor high-scoring Blimp-1 binding sites and are likely direct Blimp-1 targets  become inaccessible during terminal differentiation, even when cell cycle exit is compromised , E2F, and Yorkie in cycling tissues  binding sites that exhibit peaks of EcR binding during pupal stages according to Model Organism Encyclopedia Of DNA Elements (ModENCODE) data [Although we do not know the chromatin remodelers responsible for the closing of the distal regulatory elements at DE) data . An attrftz transcription factor 1 (ftz-f1) at the onset of metamorphosis, as well as regulating cuticle formation in the fly embryo [Blimp-1 is also highly expressed at 36-h-APF wings, following the second and highest pulse of ecdysone during metamorphosis. Blimp-1 is then immediately silenced by 44 h. We found that dynamic chromatin regions that open at 44 h are enriched for Blimp-1 binding sites. Some of these sites are potential regulatory elements for cuticle genes and other ecdysone targets such as E74EF  were heat shocked at 37°C for 42 min and then kept at 18°C. White prepupae were collected for staging and kept at 18°C until the equivalent of 12 h APF at 25°C. Then, pupae were shifted to 28°C until the equivalent of 24 h APF or 44 h APF at 25°C for dissection. Pupae develop 2.2 times more slowly at 18°C than at 25°C and 1.2 times faster at 28°C than at 25°C. All time points were adjusted to equivalent stages at 25°C for figures.Genotypes for TEM, chitin, and phosphohistone H3 (PH3) staining are as follows:w/y, w, hs-FLP; Ap-GAL4, UAS-GFP/ +; tub-gal80TS/ +w/y, w, hs-FLP; Ap-GAL4, UAS-GFP/ UAS-E2F1, UAS-DP; tub-gal80TS/ +w/y, w, hs-FLP; Ap-GAL4, UAS-GFP/ UAS-CycD, UAS-Cdk4; tub-gal80TS/ UAS-E2F1, UAS-DPCrosses were set up and kept at 18°C. White prepupae were collected and aged to the equivalent of 12 h APF and then shifted to 28°C until the equivalent of 24 h APF, 44 h APF (for PH3 staining), or 64 h APF (for TEM and chitin staining).Genotypes for Blimp-1 antibody staining are as follows:w/y, w, hs-FLP; en-GAL4, UAS-GFP/ +; tub-gal80TS/ +w/y, w, hs-FLP; en-GAL4, UAS-GFP/ UAS-CycD, UAS-Cdk4; tub-gal80TS/ UAS-E2F1, UAS-DPw/y, RNAi (BL 57479), UAS-DP; tub-gal80TS/ +w, hs-FLP; en-GAL4, UAS-GFP/ Blimp-1w/y, RNAiw, hs-FLP; en-GAL4, UAS-GFP/ +; tub-gal80TS/ whiteCrosses were set up and kept at 18°C. White prepupae were collected and shifted to 28°C until the equivalent of 36 h APF or 40–42 h APF for immunostaining.Genotypes for PCNA reporter assay are as follows:PCNA-EmGFP/ y, w, hs-FLP; +; act>CD2>gal4, UAS-RFP/+PCNA-EmGFP/ y, w, hs-FLP; UAS-E2F1, UAS-DP /+; act>CD2>gal4, UAS-RFP/+PCNA-EmGFP/ y, w, hs-FLP; +/UAS-CycD, UAS-Cdk4; act>CD2>gal4, UAS-RFP/ UAS-E2F1, UAS-DPPCNA-EmGFP line is described in [The ribed in . Crossescpr51A region, VT016704) or UAS-destabilized GFP  and incubated at 25°C. Then larval or pupal samples (staged from white prepupae) were dissected and immunostained for GFP. The cycE-GFP transgenic reporter was kindly provided by Dr. Sarah Bray [Transgenic flies were crossed with UAS-GFP . Quantification for all fluorescence images is presented in Immunostaining procedures were carried out as previously described . Primary Blimp-1 , and allTissue was incubated in Karnovsky’s fixative for at least 1 h at room temperature and then overnight at 4°C. Samples were washed with 20× volume Sorenson’s buffer 3× before postfixing in 2% osmium tetroxide in Sorenson’s buffer for 1 h at room temperature. Tissue was again washed 3× with 20× volume Sorenson’s buffer and then dehydrated through ascending concentrations of acetone and embedded in EMbed 812 epoxy resin. Semithin sections were stained with toluidine blue for tissue identification. Selected regions of interest were ultra-thin sectioned to 70 nm in thickness and poststained with uranyl acetate and Reynolds lead citrate. They were examined using a JEOL JEM-1400 Plus TEM at 80 kV. TEM images showing the phenotype are representative images. We embedded three wings for sectioning and observed the phenotype shown in all wings.S1 Figp-values. During metamorphosis, differentiation-related genes such as cuticle development are activated, whereas cell cycle genes are repressed. The underlying data for this figure can be found within (A) The heatmap shows RNA log2 fold change (compared with L3) for the indicated stages. The pattern of RNA changes during metamorphosis is separated into 18 k-means clusters. (B) Line plots of the log2 fold change versus L3 for the indicated RNA clusters. Each gene is represented by a single gray line, and the average of all genes for the given cluster is plotted in red line. GO term enrichments are also shown along with their adjusted (TIF)Click here for additional data file.S2 Fig(A) Pie charts of the proportion of dynamic peaks and static peaks for each stage examined. Peaks without significant changes (<2-fold) between neighboring time points were defined as “static.” Peaks bearing changes >2-fold were defined as “dynamic.”  Radar charts display the distribution of indicated dynamic (B) and static (C) peak categories in different distances to TSS.  Radar charts display the distribution of indicated dynamic (D) and static (E) peak categories in cds, intron, nc genes, proximal promoter (−500 bp to 150 bp of TSS), UTRs, and intergenic regions. For dynamic peaks, “closing” is defined as peaks that decrease in accessibility by >2-fold compared with the previous stage; conversely, “opening” indicates peaks that increase in accessibility by >2-fold compared with the previous stage. The underlying data for this figure can be found within (TIF)Click here for additional data file.S3 Figp-values less than 0.05. FAIRE, formaldehyde-assisted isolation of regulatory elements.Scatterplots of FAIRE peaks and corresponding genes with significant changes between two sequential stages. Significance is defined by 2-fold changes and adjusted (TIF)Click here for additional data file.S4 FigTrajectories of average changes between genes and their corresponding FAIRE peaks over the six stages for each of the 18 RNA clusters. Boxplot of the Pearson correlation coefficients between RNA and FAIRE for each RNA cluster is shown. The underlying data for this figure can be found within (TIF)Click here for additional data file.S5 FigTrajectories of average changes between FAIRE peaks and their corresponding genes over the six stages for each of the 18 FAIRE clusters. Boxplot of the Pearson correlation coefficients between RNA and FAIRE for each FAIRE cluster is shown. The underlying data for this figure can be found within (TIF)Click here for additional data file.S6 FigApterous-Gal4/Gal80ts from 12 h APF. The 24-h and 44-h wings were immunostained against PH3. (B) The number of PH3 spots of each wing is counted, and five wings for each genotype are quantified. (C) Cell cycle profile of the FAIRE samples that bypassed robust G0 by E2F/CycD/Cdk4 was examined by FACS. p-Values were determined by an unpaired t test; ****< 0.0001, ***< 0.001. The underlying data for this figure can be found within TS, temperature-sensitive Gal80; PH3, phosphohistone H3.(A) E2F or E2F/CycD/Cdk4 (bypass) was overexpressed in the dorsal layer of wing epithelia under the control of (TIF)Click here for additional data file.S7 Figp-value less than 0.05 are labeled in red. Cdk, cyclin-dependent kinase; CycD, Cyclin D; E2F, E2F transcription factor; FAIRE, formaldehyde-assisted isolation of regulatory elements; FAIRE-seq, FAIRE sequencing; MA plot, scatter plot onto M (log ratio) and A (mean average) scales; RNA-seq, RNA sequencing.MA plots of RNA (A) and FAIRE (B) changes of 24- and 44-h wings compared with control. Genes and peaks that are significant in changes with 2-fold difference and adjusted (TIF)Click here for additional data file.S8 Fig The heatmap shows the temporal dynamics during normal development for the peaks that are more accessible or less accessible at 44-h wings expressing E2F/CycD/Cdk4, plotted as a fraction of the maximum FAIRE rpkm value. Compromising G0 leads to the failure of proper closing of 36-h peaks as well as delayed opening of 44-h peaks. (C) Overlap between peaks that normally open at 36 h in wild-type and peaks more accessible at 44 h bypassed wings. (D) Overlap between peaks that normally open at 44 h in wild-type and peaks less accessible at 44 h bypassed wings. The underlying data for this figure can be found within (TIF)Click here for additional data file.S9 FigE74EF and ftz-f1 loci with Blimp-1 binding sites are shown. (D) Expression changes of Blimp-1, ftz-f1, and E74EF during normal development. The underlying data for this figure can be found within (A) The Blimp-1 motif is enriched in the dynamic peaks disrupted by E2F or bypass found by AME analysis. (B) A list of genes containing peaks that fail to open at 44 h with high-scoring Blimp-1 binding sites. (C) Chromatin accessibility changes at (TIF)Click here for additional data file.S10 FigBlimp-1. (B) Expressing Blimp-1RNAi in the posterior wings by engrailed-Gal4/Gal80TS from 0 h APF reduces the level of Blimp-1 protein at 36-h wings. APF, after puparium formation; Gal80TS, temperature-sensitive Gal80; Blimp-1RNAi, RNA interference against Blimp-1.(A) Blimp-1 antibody staining in wild-type L3, 6-h, and 36-h wings corresponds to the gene expression changes of (TIF)Click here for additional data file.S11 Figp-value: <0.0001, stg p-value: <0.0001, e2f1 p-value: 0.0232, cycE p-value: 0.0005). (B) Chitin staining was compared between the dorsal and ventral epithelium for each wing, and the ratio was calculated with wild-type control wings set to 1. N = 3–5 wings for each genotype. Chitin signal is significantly affected by manipulating cell cycle exit . (C) Blimp-1 staining intensity was compared between posterior and anterior compartments of each wing, and the ratio (P:A) was calculated. N = 3–4 wings for each genotype. Bypassing cell cycle exit significantly delays the temporal regulation of Blimp-1 protein . The underlying data for this figure can be found within This figure provides quantification for GFP reporters in (TIF)Click here for additional data file.S1 TableFAIRE, formaldehyde-assisted isolation of regulatory elements; RPKM, reads per kilobase of transcript, per million mapped reads.(XLSX)Click here for additional data file.S2 TableRNA-seq, RNA sequencing; RPKM, reads per kilobase of transcript, per million mapped reads.(XLSX)Click here for additional data file.S3 TableRNA-seq, RNA sequencing.(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.


Estrogen receptor α (ERα) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcriptional events that promote tumor growth. ERα binding activity largely depends on access to binding sites on chromatin, which is facilitated in part by Pioneer Factors (PFs). Transcription factors operate in complexes through thousands of genomic binding sites in a combinatorial fashion to control the expression of genes. However, the extent of crosstalk and cooperation between ERα pioneer factors and more collaborative transcription factors in breast cancer still remains to be elucidated systematically.Methods: Here, we determined the genomic binding information of 40 transcription-related factors and histone modifications with ChIP-seq in ENCODE and integrated it with other genomic information , forming a multi-dimension network to illuminate ERα associated transcription.Results: We show that transcription factor, NR2F2 binds to most sites independently of estrogen. Perturbation of NR2F2 expression decreases ERα DNA binding, chromatin openning, and estrogen-dependent cell growth. In the genome-wide analysis, we show that most binding events of NR2F2 and known pioneer factors FOXA1, GATA3 occur together, covering 85% of the ERα binding sites. Regions bound by all the three TFs appeared to be the most active, to have the strongest ERα binding and to be enriched for the super enhancers.Conclusions: The ERα binds to pre-accessible sites containing ERE elements bound by the three transcription factors .The three genes were also identified to correlate with decreased metastatic potential in patient cohorts and co-regulate each other. Together, our results suggest that NR2F2 is a cofactor with FOXA1 and GATA3 in ERα-mediated transcription. It's becoming increasingly clear that transcription is mediated by a limited cohort of transcription factors with combinatorial fashion to form transcription complex and organize the large diversity of gene-expression patterns to the linage commitment cell types This transcription factor colocalization and complexes at enhancer elements govern the genomic circuitry, and these can greatly influence on chromatin accessibility and transcription programs in vivoWe have utilized genome-wide analysis of ChIP-seq data of more than 30 transcription factors/histone modification markers in ER positive breast cancer model cell line MCF-7, from ENCODE (The Encyclopedia of DNA Elements) The MCF-7, ZR-75-1, T-47D, MDA-MB-453, SKBR3, SUM159, MDA-MB-435, MDA-MB-231 and LM-2 breast cancer cell lines have been utilized in this study. Cell culture conditions is provided in the standard procedures.Lentiviruses were produced by transient transfection of 293T cells with pHAGE, psPAX2, and pMD2.G plasmids. The virus containing medium and polybrene  were added to MCF-7 cells. Infected cells were selected with 0.5 μg/mL puromycin. Only early passage cells were used in all experiments. Cells expressing either GFP or the tag alone (including the TEV protease recognition site) were used as a control cell line.ChIP experiments have been performed as per standard protocols Data was analyzed and processed by a unified procedure to eliminate the bias by data processing   is widely used to map chromatin accessibility genome-wide Covalent modifications are a main chromatin property Immunofluorescence assays against NR2F2 in MCF-7 breast cancer cells deprived of oestrogen further demonstrated its localization to the nucleus Figure H. AlthouWe separately calculated the overlap degree of ERα with NR2F2, GATA3, FOXA1. However, what percentage of ER binding events are synergistically covered by NR2F2, GATA3, FOXA1 remains to be investigated. Thus, we calculated to what degree specific ER-binding regions used one, many or none of the GATA3, NR2F2, and FOXA1. The pie chart showed that 25% of all ER-binding events were bound by all three factors, 36% were bound by two of the the three factors and 24% were bound by one of the three  obviously showed that these TFs were significantly co-expressed with ERα (co-expression coefficient=0.88) and other hormone receptors, such as the PR and AR, through unsupervised clustering analysis  to DNA. We collected methylation 450K microarrays for more than one thousand breast cancer patients from TCGA (The Cancer Genome Atlas). After filtering the data, we retained 773 effective patients with more than 40 methylation sites across the promoters of the three genes. Unsupervised free clustering showed that the CpG sites in the promoters of these three genes tended to have relatively low methylation levels in ER positive breast cancer and hypermethylation in ER negative patients. The hypermethylation in promoter regions suppressed binding of the transcription factors, including POLII, and thus inactivated transcription of the three TFs Figure C. Our reER interact with numerous associated proteins that can help to tether the receptor to DNA including pioneer factor and that can keep optimal open DNA and that can possess intrinsic enzymatic activity. Here, by screening the interactome of the ER and other hormone receptors to explore candidate interactions, we found NR2F2 is required for growth and binds to most sites independently of estrogen. NR2F2 is required for ER binding and inhibition of this protein decreases ATAC-seq signal. NR2F2 acts as a functional co-factor of ERα cobinding with pioneer factor FOXA1 and GATA3 by binding ER-related enhancers. In addition, we demonstrated that sites occupied by all three TFs were associated with the highest chromatin opening and ER recruitment degrees. NR2F2, GATA3, and FOXA1 jointly cover 85% of ER binding sites.de novo established accessible chromatin to drive the complement of the ERα core function at short time.The sequential binding of NR2F2, GATA3, and FOXA1 in ER binding events is still undetermined. Previously, it was reported that GATA3 acts upstream of FOXA1 when mediating ESR1 by shaping the enhancer accessibility The expression of these three transcription factors can mostly define the positive or negative ER status. Low levels of these three TFs were strongly associated with an ER negative status, PR-negative status, poor survival, positive lymph node status, higher histological grade, and increased risk for recurrence and metastasis. They also had high expression levels in model ER-AR+ cell line MDA-MB-453 cells. Our data shown that AR can function as prognostic targets in ER negative patients but has no effect on ER positive ones Figure B and S2CAmong these three factors, FOXA1 and GATA3 are important transcription regulatory proteins and known ER-interacting transcription factors. GATA3 is reported to be highly expressed in the mammary epithelium and is expressed exclusively in the luminal epithelial cell population, where it specifies and maintains luminal cell differentiation. Inactivation of GATA3 results in failure of the mesenchymal-epithelial transition (MET), which increases breast cancer metastasis.Epigenetic silencing of tumour suppressor genes by DNA methylation is a hallmark of tumours Taken together, our results show that the interplay of cofactor NR2F2 with pioneer factors GATA3 and FOXA1, which promote an open chromatin structure, subsequent nuclear receptor binding, and resultant initiation of E2-induced transcriptional programmes, are essential for optimal ER binding to the DNA. We further uncovered the functional importance of an enhanceosome that exerted significant combinatorial control over the transcriptional network related to proliferation and metastasis of ER positive breast cancer cells.Supplementary figures and supplementary table 1.Click here for additional data file.Supplementary table 2.Click here for additional data file.Supplementary table 3.Click here for additional data file.Supplementary table 4.Click here for additional data file.Supplementary table 5.Click here for additional data file.


EphB4 gene encodes for a transmembrane tyrosine kinase receptor involved in embryonic blood vessel differentiation and cancer development. Although EphB4 is known to be regulated at the post-translational level, little is known about its gene regulation. The present study describes the core promoter elements’ identification and cloning, the cis-regulatory elements’ mapping and the serum regulation of the human EphB4 gene promoter region. Using bioinformatic analysis, Sanger sequencing and recombinant DNA technology, we analyzed the EphB4 gene upstream region spanning +40/−1509 from the actual transcription start site (TSS) and proved it to be a TATA-less gene promoter with dispersed regulatory elements characterized by a novel motif-of-ten element (MTE) at positions +18/+28, and a DPE-like motif and a DPE-like-repeated motif (DRM) spanning nt +27/+30 and +32 +35, respectively. We also mapped both proximal (multiple Sp1) and distal (HoxA9) trans-activating/dispersed cis-acting transcription factor (TF)-binding elements on the region we studied and used a transient transfection reporter assay to characterize its regulation by serum and IGF-II using EphB4 promoter deletion constructs with or without the identified new DNA-binding elements. Altogether, these findings shed new light on the human EphB4 promoter structure and regulation, suggesting mechanistic features conserved among Pol-II TATA-less genes phylogenetically shared from Drosophila to Human genomes.The  EphB4 gene’s regulatory region, seek unidentified promoter elements and provide a widely usable model on the structure, functional map and serum regulation of the EphB4 human gene promoter.EphB4 is a member of the Eph/Ephrin tyrosine receptor family involved in the formation of blood vessels during embryonal development . It is tRestriction enzymes and DNA-end modifying enzymes used for cloning were purchased from New England Biolabs . All other molecular biology reagents, unless specified, were purchased from Sigma . Synthetic oligos used for + 40/+10 core promoter element reconstitution and cloning were purchased from IDT .A 1509 nt PCR fragment containing the genomic regions corresponding to +7/−1509  was sequenced using Sanger method from a pGL3-Luc reporter plasmid provided by C. Urbich  using GL2primer2 and RVprimer3, respectively. Chromosomal consensus at the genomic DNA level, a transcription factor (TF) motif search and motif/domain comparisons were all performed using nBlast (NCBI).EphB4) promoter fragment(s) in a BamHI-KpnI fragment, and a KpnI-XhoI pGL3-luc plasmid backbone fragment along with a synthetic 54 nt synthetic reconstructed fragment spanning EphB4 upstream promoter region from position +40 site from inr A+ position in a BamHI-XhoI frame. This last fragment was the result itself, of a double-synthetic, oligo pair  and colony screening. Successful inclusion of the +40/–10 promoter region cloning was confirmed in the plasmid DNA extracted from selected positive colonies initially screened by NcoI double digest (since a NcoI RE site is present between Inr and motif-of-ten element (MTE)/DPE in the EphB4 native core promoter sequence) followed by Sanger sequencing. As a result of the upstream cloning sequencing, two types of EphB4 promoter constructs either containing the DPE or MTE cluster were generated, two of which ended at downstream position −1028 and two at position −1509, these last two containing a previously described HoxA9-binding region (see EphB4 promoter constructs described in this study).The +40/−1509 and +40/−1028 constructs were obtained by triple ligation reaction with sticky-ends fragments containing, respectively, the original −7 (BamHI)/1509 (or the −7/−1028 A) under puromycin-selective conditions were transiently transfected with the EphB4 promoter constructs described herein. Following transfection in low serum optimized medium , cells were grown to sub-confluence and then serum starved for 16 h before stimulation by replacing the serum free media with either 10% containing media  or serum free media containing 0.1% bovine serum albumin and 10 nM IGF-II  for following 16 h prior harvesting the cells in Luciferase assay buffer . The Luciferase reporter Dual-GloTM and Bright-GloTM kits and reagents used throughout the study were purchased by Promega. Results from luciferase assay are represented as means ± standard errors of the means (SEMs), and statistical analysis was by a Student’s t-test using unequal variance.MEFs with a null mutation for the IGF1R (R-cells) constitutively expressing either the human IGF1R (R+) or the insulin receptor isoform A  was used for a bioinformatic search for known transcription factors binding elements. This initial search did not result in the identification of canonical TATA box motifs spanning region −40 nt to +40 from the transcription start site (TSS), nor to the identification of the actual Inr sequence or other known core promoter elements within the previously cloned genomic fragments [homeobox A9 (HoxA9) regulatory motif whose mutation was able to ablate serum-induced transcription [EphB4 gene expression. However, in the absence of known core promoter elements within the previously cloned genomic region, we decided to investigate further. Therefore, we plotted the sequence spanning −7/−1509 in EphB4’s upstream flanking region against the chromosomal genomic context on chromosome 7 (GRCh37:7:100355490:100525847:1) obtained from the Ensemb database. Furthermore, in order to identify or rule out potential previously undetected core transcriptional elements, we restricted our bioinformatic search on the region comprised between the 5′ end of the EphB4 transcribed gene and the transcription (A+1) start site which we identified in the previously invoked position  [EphB4 core promoter elements on the overall transcriptional regulation of this gene, we decided to clone the missing +40/−6 regulatory sequence into two formerly described core promoter-deficient constructs (kind gift of C. Urbich) to reconstitute the regions spanning +40/−1509 and +40/−1028, respectively. The two novel Inr/MTE/DPE-like bearing constructs were cloned as described in Methods and EphB4 promoter constructs were generated either containing the Inr/MTE/DPE-like core elements or not, two of which ended at downstream position −1028 (containing multiple Sp1 motifs spanning −60/−528) and two at position −1509 (containing both multiple Sp1 and HoxA9-binding elements). A graphic summary of the EphB4 promoter constructs and their known transcriptional regulatory elements is provided in In order to identify potential growth-factor-responsive elements in the human ragments . Nonethecription . This fiposition . This anposition A,B reveapromoter B allowedDrosophila genes, we decided to perform a comparative/consensus analysis between the novel MTE sequence in the human EphB4 promoter and the previously reported Drosophila MTE sequences from Lim et al. [Drosophila MTE-bearing genes might be compensated by the synergism between adjacent MTE and DPE elements towards transcription initiation, as observed in other of Pol-II-regulated genes [EphB4 MTE/DPE motifs in the EphB4 promoter with the suggested consensus sequences of Thiesen et al. [EphB4 downstream promoter along with a full shared consensus (A) in position +28. Of interest in the EphB4, DPE-like downstream region is the presence of a DPE-like repeated GACG motif (DRM) at position +32/+35. It will be useful to determine the physical interaction features with the Pol-II core transcriptional machinery conferred by this new type of core promoter element configuration.Since the MTE was first described in a number of m et al. , as showed genes . Theisened genes  characteed genes  conveyedn et al.  and Kadon et al.  to exertEphB4 promoter transcriptional activation by serum, as described under methods. As summarized in p ≤ 0.0001) with the EphB4 promoter construct +40/−1028 bearing the identified Inr/MTE/DPE-like downstream elements and the defect of the region bearing the HoxA9 transactivation motif at −1375. This result suggests an underscored role for the multiple proximal Sp1-binding elements in driving EphB4 serum-stimulated transcription. This result is also consistent with the well-known synergistic ability of multiple Sp1 complexes to induce the transcription of TATA-less genes due to the efficient recruitment of TAF4 and TAF7 (part of TFIID) towards transcription initiation, an effect known as Sp1 super-activation (reviewed by Wierstra [Sp1+HoxA9) are present, provided only a discrete activation suggesting a competitive or negative cooperative role of Sp1 and HoxA9 (compare +40/−1509 with +40/−1028). When looking at the serum-response observed with the EphB4 core-promoter (Inr/MTE/DPE-like)-deficient constructs (−7/−1509 and −7/−1028), our results are consistent with the results previously obtained by Bruhl et al. [HoxA9 distant motif showing the most effective transactivation activity towards serum-stimulated EphB4 transcription. On the other hand, the isolated proximal Sp1-binding elements-spanning region −60/−528 (contained in the −7/−1028 construct) did not appear to play a major role in EphB4 serum-dependent transcription in absence of the Inr/MTE/DPE-like core elements. To explain the transcriptional activity that both we . Under the studied conditions, IGF-II stimulation (10nM) did not exert transcriptional effects (not significant at p > 0.6 for both constructs) similar to those observed with serum, although a minimum effect was observed in Sp1+/HoxA9 motif-less promoters, irrespective of the presence of the Inr /MTE/DPE-like downstream elements (+40/−1028 and −7/1028)  .II60/40 subcomplex or as part of TFIID) located about 30 nucleotides downstream of the transcription start site of many TATA-box-deficient (TATA-less) promoters from Drosophila and humans [Drosophila, about 26% of promoters are TATA-less and bear a DPE motif [EphB4 MTE-DPE-like module at these positions might support a scenario with similar core promoter interactions. However, the DPE-like repeat (GACG) we found in the EphB4 downstream promoter at positions +27/+30 and +32/+35 , separated by one “A” nt bridge in position +31, is in apparent contrast with the classic DPE motif spanning positions +28/+32 [EphB4 gene regulation and for other TATA-less genes bearing similar downstream core elements. We recently found IGF-II to be a key driver of protein EphB4 ectopic expression in malignant mesothelioma cells and we identified a ubiquitin/UPS-associated post-transcriptional mechanism tightly regulating EphB4 protein degradation/stabilization [EphB4 promoter by IGF-II compared to serum in R-IRA cells would therefore suggest that IGF-II is not a major driver of the EphB4 gene transcriptional activation and supports our previous finding that the strong dependence of EphB4 protein expression by IGF-II is linked to a post-transcriptional mechanism. Indeed, the conclusion in which we claim that IGF-II is not a major driver of EphB4 transcriptional activation is based upon the fact that IGF-II is a major serum component. Therefore, the difference observed in transcriptional activation between serum and IGF-II on the studied regulatory promoter regions is strong evidence that other growth factors than IGF-II must be responsible for the serum effects observed under the conditions tested. Nonetheless, we cannot rule out an IGF-II specific role in the EphB4 gene’s transcriptional regulation via far distant elements and/or under different contextual cellular events  not tested in our present study.The core promoter in human genes is the region from −40 to +40 and flanks the transcription start site (TSS) at A+1 . There ad humans . In DrosPE motif . FurtherPE motif . AlthougPE motif . TheisenPE motif  found th +28/+32 . This fiEphB4 gene promoter to be a serum-responsive TATA-less Inr/MTE/DPE-like-driven promoter. Our findings support a synergistic role of the multiple Sp1 motif containing proximal region with the identified Inr/MTE/DPE-like core promoter elements for exerting a full serum transcriptional response. Furthermore, our study shows that serum-dependent EphB4 transcriptional regulation is affected by a negative cooperative effect between proximal and distal regulatory elements; namely, between the upstream proximal promoter region containing multiple Sp1-binding motifs (−60/−528) and the distant promoter region bearing a HoxA9-binding motif (at −1375), respectively. Further studies will need to clarify the mechanistic and regulatory aspects linked to the identified EphB4 downstream promoter elements and the competing contextual roles of this promoter cis-acting elements at the proximal and distal sites.In summary, using DNA sequencing, recombinant DNA cloning, bioinformatics and a gene reporter assay, here, we reported the human


IRF1 can trigger the production of endogenous interferon and the expression of ISGs, suggesting that IRF1 can positively regulate IFN signalling. Importantly, the mRNA and protein levels of IRF1 were reduced upon FCV infection, which may be a new strategy for FCV to evade the innate immune system. Finally, the antiviral activity of IRF1 against feline panleukopenia virus, feline herpesvirus, and feline infectious peritonitis virus was demonstrated. These data indicate that feline IRF1 plays an important role in regulating the host type I IFN response and inhibiting feline viral infections.Interferons (IFNs) can inhibit most, if not all, viral infections by eliciting the transcription of hundreds of interferon-stimulated genes (ISGs). Feline calicivirus (FCV) is a highly contagious pathogen of cats and a surrogate for Norwalk virus. Interferon efficiently inhibits the replication of FCV, but the mechanism of the antiviral activity is poorly understood. Here, we evaluated the anti-FCV activity of ten ISGs, whose antiviral activities were previously reported. The results showed that interferon regulatory factor 1 (IRF1) can significantly inhibit the replication of FCV, whereas the other ISGs tested in this study failed. Further, we found that IRF1 was localized in the nucleus and efficiently activated IFN- Feline calicivirus (FCV) is a highly contagious pathogen of cats and usually causes mild to serious oral and upper respiratory tract disease . FCV belβ by preventing IRF3 activation [β, but other FCV strains failed [Only recently was the Felis catus genome sequenced, so little is known about feline innate immunity and the interaction of FCV with the feline innate immunity, which limits the understanding of the pathogenesis of FCV and other feline viruses . The nontivation . Meanwhis failed . In addis failed .β can suppress the infection of FCV. Treatment with these IFNs reduced the viral yield of FCV F9 [ω has been marketed in Japan  and is used to treat feline calicivirus and canine parvovirus infections [β can efficiently inhibit the replication of the FCV 2280 strain. Interferons (IFNs) are a component of an early response to invading pathogens and induce the expression of hundreds of IFN-stimulated genes (ISGs) [β [α, Tetherin, and MX2 [FCV is very sensitive to interferons, and IFN-f FCV F9 . Feline fections , 12. We s (ISGs) . The mecs (ISGs) . InterfeISGs) [β . ISG20 iISGs) [β . InterfeISGs) [β . SAMHD1 ISGs) [β  and restISGs) [β . In addiWhile FCV is sensitive to interferons, the ISGs that could inhibit the replication of feline calicivirus have not been identified. Here, we evaluated the antiviral activity of ten feline ISGs  against FCV using a transient transfection method and found that feline IRF1 could efficiently suppress the replication of FCV. Further, we found that feline IRF1 can trigger the production of endogenous interferon and the expression of ISGs, suggesting that IRF1 can positively regulate IFN signalling. Importantly, the mRNA and protein levels of IRF1 were reduced upon FCV infection, which may be a new strategy for FCV to evade the innate immune system. Finally, the antiviral activity of IRF1 against feline panleukopenia virus, feline herpesvirus, and feline infectious peritonitis virus was also demonstrated.μg/ml streptomycin. FCV strain F9, FHV-1 strain HR-1, FPV strain VR-638, and FIPV strain 2034 were propagated and titred in CRFK cells.Crandell Rees feline kidney (CRFK) cells were maintained in Dulbecco's modified minimum essential medium (DMEM) (Gibco) containing 10% foetal bovine serum , 100 U/ml penicillin, and 100 β encodes the Flag-tagged feline IFN-β and the plasmid pIFN-β-Luc contains a luciferase (Luc) expression cassette driven by the feline IFN-β promoter as described previously [The p3×Flag-IRF1, p3×Flag-RNase L, p3×Flag-SAMHD, p3×Flag-ISG15, p3×FlagISG20, p3×Flag-IFIT3, p3×Flag-MX-1, p3×Flag-GBP6, p3×Flag-OASL and p3×Flag-IRF2 plasmids express Flag-tagged feline IRF1 (Accession number: XM_003980752.4), RNase L (Accession number: XM_006942762.2), SAMHD (Accession number: XM_003983547.3), ISG15 (Accession number: NM_001130843.2), ISG20 (Accession number: XM_019831901.1), IFIT3 (Accession number: XM_011287196.2), MX-1 (Accession number: XM_006935851.2), GBP6 (Accession number: XM_003990288.3), OASL (Accession number: XM_003994734.3), and IRF2 (Accession number: XM_019828250.1), respectively. The pMyc-IRF1 plasmid expresses IRF1 with a Myc-tag. The plasmid p3×Flag-IFN-4/well) grown in 48-well plates were cotransfected with 0.5 μg/well reporter plasmid, 0.02 μg/well plasmid pRL-TK (Promega) , or the indicated expression plasmid. Cells were infected with SeV (100 haemagglutinating activity units/well) at 12 h after cotransfection. The cells were lysed at 8-10 h postinfection, and the firefly and Renilla luciferase activities were measured with the dual-luciferase reporter assay system (Promega), according to the manufacturer's protocol. Relative luciferase activity in each sample was determined using the ratio between the activities of firefly and Renilla luciferases.The protocol for this luciferase assay has been described previously . Briefly5/well) were seeded into 24-well culture plates for 24 h and then were transfected with 1 μg of ISG expression plasmids or mock-transfected with 1 μg of p3×Flag-CMV-10. FCV strain F9 with an MOI of 0.01 was inoculated into cells at 24 h after transfection. The cell supernatant and cells were harvested for the examination of viral titres and viral RNA, respectively, at 24 h postinfection.Briefly, CRFK cells  using FITC-conjugated anti-canine parvovirus monoclonal antibody .Cells were washed with cold PBS and lysed with RIPA Lysis Buffer  with 0.1 mM PMSF, and then lysates were cleared by centrifugation at 12,000 g for 5 min at 4°C. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% skim milk for 1-2 h at room temperature and then incubated for 1 hour at room temperature with mouse anti-FLAG M2 MAb , rabbit anti-Myc polyclonal antibody , rabbit anti-IRF1 monoclonal antibody , rabbit anti-GADPH antibody , and mouse anti-FCV VP1 monoclonal antibody (made by our laboratory).After three rinses in TBST buffer, the membranes were incubated at room temperature for 1 h with IRDye 800DX conjugated anti-rabbit IgG or IRDye 800-conjugated anti-mouse IgG  diluted with TBST as a secondary antibody. After the third wash, membranes were visualized and analysed with an Odyssey infrared imaging system (LI-COR Biosciences). t-test and one-way ANOVA with Prism 5.0 software (GraphPad Software). A p value of <0.05 was chosen to indicate significance.The significant differences between the experimental groups were determined with the pairedβ (positive control), IRF1, RNase L, and SAMHD significantly reduced viral RNA levels compared with those of the vector transfection group  encoding the DNA-binding domain (DBD) are structurally and functionally conserved among the IRF family of proteins . We analTo explore the subcellular localization of fe-IRF1, the fe-IRF1 CDS were inserted into pEGFP-N1 and named pEGFP-IRF1. Confocal microscopy indicated that the fe-IRF1 fusion protein was localized in the nucleus, which is the same subcellular localization as other IRF1s.β signalling pathway, CRFK cells were cotransfected with the fe-IRF1 plasmid and IFN-β , IFITM1 (40-fold), and Viperin (300-fold) , feline infectious peritonitis virus (FIPV), and feline panleukopenia virus (FPV). At 48 h postinoculation, the viral yields were tested. The results indicated that overexpression of fe-IRF1 can efficiently impede the replication of FPV , FIPV F, and FHVβ gene [The IFN system has a profound role in inducing the expression of antiviral proteins encoded by interferon-stimulated genes . The proβ gene . More imβ promoter and found to be constitutively expressed at a low basal level in most cell types [IRF1 is a transcription factor that regulates the innate and adaptive immune responses . The IRFll types . The idell types , 33, butll types .β and ISRE promoter by its DBD domain, which can upregulate the expression of many genes such as IFN-α/β and many ISG genes such as 2′, 5′-OAS, protein kinase R[The murine IRF1 contains two nuclear localization signal sequences (NLS), 120RKERKSK and 132KSKTKRK. A 24-amino acid sequence that contains both sequences was found to mediate nuclear translocation of IRF1 . The N-t kinase R, and Vip kinase R. It is eIRF1 mRNA rises in response to IFNs, double-stranded RNA (dsRNA), cytokines, some hormones , and virIn conclusion, we demonstrate for the first time that fe-IRF1 inhibits the replication of FCV and other feline viruses. We have also provided evidence that FCV infection suppresses the expression of IRF1 by reducing the level of fe-IRF1 mRNA, which highlights a potential immune evasion strategy for FCV.