What is more, interfering with this process is an innovative strategy to overcome the chemo‐resistance of GBM cells.Glioblastoma multiforme (GBM) is a primary tumour of the central nervous system (CNS) that exhibits the highest degree of malignancy. Radiotherapy and chemotherapy are essential to prolong the survival time of patients. However, clinical work has demonstrated that sensitivity of GBM to chemotherapy decreases with time. The phenomenon of multi‐drug resistance (MDR) reminds us that there may exist some fundamental mechanisms in the process of chemo‐resistance. We tried to explore the mechanism of GBM chemo‐resistance from the perspective of energy metabolism. First, we found that the oxidative phosphorylation (OXPHOS) level of SHG44 and U87 cells increased under TMZ treatment. In further studies, it was found that the expression of PINK1 and mitophagy flux downstream was downregulated in GBM cells, which were secondary to the upregulation of TP53 in tumour cells under TMZ treatment. At the same time, we examined the mitochondrial morphology in tumour cells and found that the size of mitochondria in tumour cells increased under the treatment of TMZ, which originated from the regulation of AMPK on the subcellular localization of Drp1 under the condition of unbalanced energy supply and demand in tumour cells. The accumulation of mitochondrial mass and the optimization of mitochondrial quality accounted for the increased oxidative phosphorylation, and interruption of the mitochondrial fusion process downregulated the efficiency of oxidative phosphorylation and sensitized GBM cells to TMZ, which was also confirmed in the  The progression‐free survival of patients with this condition after diagnosis is 6.2–7.5 months; the median survival is 14.6–16.7 monthsIn the studies of the chemo‐resistance, mitochondria are under spotlight for a long time. Mitochondria are involved in vital processes such as energy synthesis, regulation of calcium ion, ROS generation and apoptosis executionAs we all know, TMZ kills tumour cells by inducing DNA damage, and the survival cells are always with upregulated DNA damage response or multiple stress responses. Although these responses vary, energy support is essential.22.1TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N‐Acetyl‐L‐cysteine (NAC) were purchased from Sigma‐Aldrich. MitoTracker™ Red FM (M22425), Hoechst 33342 (H1399) and anti‐Ubiquitin WB Antibody (13–1600) were purchased from Invitrogen  . Anti‐phospho‐Ubiquitin (Ser65) (ABS1513‐I) was purchased from Merck KGaA company (1:1000). Anti‐P53 (21891–1‐AP), anti‐PINK1 (23274–1‐AP), anti‐Parkin (14060–1‐AP), anti‐Drp1 (12957–1‐AP), anti‐Opa1 (27733–1‐AP), anti‐Mfn1 (13798–1‐AP), anti‐Mfn2 (12186–1‐AP), anti‐Caspase‐9 (10380–1‐AP), anti‐BAX (50599–2‐Ig), anti‐Caspase‐3 (19677–1‐AP), anti‐VDAC1 (10866–1‐AP), anti‐Lamin B (12987–1‐AP), anti‐Cytochrome c (10993–1‐AP), and anti‐β‐actin (60008–1‐Ig) were purchased from ProteinTech Group, Inc.,  . Anti‐γ‐H2A.X (ab81299) was purchased from Abcam (1:1000). Anti‐AMPKα (2532) and p‐AMPKα (50081) were purchased from Cell Signaling Technology, Inc.  (1:1000). Anti‐phospho‐DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti‐phospho‐DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. .2.22 atmosphere.Human glioblastoma cell lines  were obtained from the Chinese Academy of Medical Sciences and cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, penicillin (50 U/ml) and streptomycin (50 µg/ml) purchased from Life Technologies and incubated at 37°C in a 5% CO2.36 U87MG cells were suspended in 100 µl of phosphate buffer saline (PBS) (Beyotime Institute of Biotechnology) and injected subcutaneously into the right armpit of the nude mice. The animals were raised in a specific pathogen‐free (SPF) environment, and tumour volume was observed regularly. One week later, when the tumour volume reached 50 mm3, the nude mice were randomly divided into four groups, with eight mice in each group. The treatment conditions for each group were as follows: control group: 200 µl of 5% Carboxymethyl Cellulose (CMC)—daily gavage; TMZ group: 40 mg of TMZ suspended in 200 µl of 5% CMC—daily gavage; WY14643 group: 10 mg of WY14643 suspended in 200 µl of 5% CMC—daily gavage; TMZ+WY14641 group: TMZ (40 mg) + WY14643 (10 mg) suspended in 200 µl of 5% CMC—daily gavage. The status of the nude mice was observed daily. Mice were weighed and the size of each tumour was measured every 2 days. The experiment was terminated when the longest diameter of the tumour reached 2 cm or signs of ulceration appeared. Upon termination of the experiment, the nude mice were sacrificed, and the tumour was stripped, photographed and weighed. The tumour tissue was used for the following experimental tests: (1) evaluating the ATP content of tissue cells; (2) the total protein was extracted from the tumour tissue for Western blotting (WB); (3) part of the tumour tissue was stored in 2.5% dialdehyde and analysed by transmission electron microscopy; and (4) part of the tumour tissue was stored in 4% paraformaldehyde for immunohistochemical analysis.The experimental protocol was approved by the local ethics committee (20200921–1). Nude mice were adapted to the experimental environment for a week prior to experimentation. Then, 5 × 102.4Cells were lysed in Prusiner's buffer , EDTA (5 mM), Triton ×‐100 (0.5%), and deoxycholate and protease inhibitor cocktail). The homogenates obtained were then briefly sonicated. Aliquots of 10µg of total protein were then loaded onto 8%–16% SDS‐PAGE gels. After migration, proteins were wet‐transferred to PVDF membranes and immunoblotted using certain antibodies. Immunological complexes were detected with either anti‐rabbit or anti‐mouse IgG‐coupled peroxidase antibodies  by the electrochemiluminescence detection method (Roche Diagnostics S.A.S).2.5Cells were grown on coverslips in 24‐well plates and treated with different agents. After staining with MitoTracker for 30 min, the coverslips were rinsed with PBS and fixed with 4% (w/v) paraformaldehyde for 25 min at room temperature followed by a PBS rinse. Permeabilization with 0.1% (v/v) Triton ×‐100 for 7 min was followed by blockade with 10% horse serum for another 30 min. Following incubation with primary antibodies (1:100) at 4°C for 12 h, the coverslips were incubated with fluorescent secondary antibodies (1:200) for 30 min, rinsed with cold PBS and stained with Hoechst 33342 in accordance with the manufacturer's instructions for 7min before a final PBS rinse. A Revolve Hybrid Microscope (Discover ECHO) was used for image capture. Images were further processed by ImageJ Software version 1.52 s  to assess the intensity of immunofluorescence (IF), as well as the mitochondrial parameters. For the evaluation of mitochondrial fusion, we evaluated three images in each treatment group from three independent experiments using ImageJ Software. Two parameters were used to assess the level of mitochondrial fusion: the mean size of the mitochondrial network (MS) and the mean length of mitochondria (ML).2.65 cells/well. Cells were then incubated overnight at 37°C, and the medium was replaced with fresh complete medium. After 24 h, the culture medium was collected and proteins were extracted by sonication. Extracted proteins were then quantified with a Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Then, we measured the concentrations of glucose and lactate with glucose (RsBio) and lactate assay kits (Jiancheng Bio), respectively. The glucose consumption in each experimental group was calculated as follows: Glucose consumption = glucose concentration (fresh complete medium)–glucose concentration .Cells were seeded in 6‐well plates at a density of 3 × 102.75 cells/well. Following overnight incubation at 37°C, the medium was replaced with fresh culture medium. After 6 or 24 h, in accordance with the manufacturer's instructions, cells were washed with PBS, and then, their ATP levels were determined using an ATP Assay Kit (Beyotime Institute of Biotechnology).Cells were seeded in 6‐well plates at a density of 5 × 102.8® Xtra‐Oxygen Consumption Assay (Luxcel Biosciences Cork) using a CLARIOstar microplate reader (BMG Labtech). In brief, cells were plated at a density of 8 × 104 cells/well in 96‐well plates  and allowed to adhere overnight at 37°C using a plate block heater. The culture medium was removed from all wells and replaced with 160 μl of pre‐warmed reaction mixture, including 10 μl of reconstituted MitoXpress® Xtra reagent, 150 μl of fresh culture media with TMZ  in each well. The wells were sealed by adding two drops of pre‐warmed HS Mineral Oil, and the plate was immediately analysed in a microplate reader.Cellular oxygen consumption rate (OCR) was measured by the MitoXpress2.94 cells per well. After exposure to TMZ and/or Mdivi1 or WY14643, for 24 h, the cells in each well were incubated with MTT solution (0.5 mg/ml) dissolved in PBS for 4 h at 37°C. Then, 150 µl of DMSO was added to each well. The absorbance was measured at 570 nm using a CLARIOstar microplate reader (BMG Labtech). Cell viability was then calculated as follows: cell viability = absorbance of experimental group/absorbance of control group × 100%.Cells were seeded in 96‐well plates with 100µl of complete DMEM medium at a density of 1 × 102.10RNA was extracted using a RNeasy kit (Qiagen) in accordance with the manufacturer's protocol. Quantitative real‐time reverse transcription‐PCR (qRT‐PCR) was then performed using a two‐step method. Data analysis was based on the delt‐delt‐Ct method using β‐Actin as a normalization control. PCR was conducted in a QuantStudio5 real‐time PCR system (Thermo Fisher) and analysed using QuantStudio Design & Analysis software v1.3.1 (Thermo Fisher).In order to evaluate the mtDNA copy number, we extracted DNA from cells in each group and determined the copy number of the mtDNA gene ND1, as normalized to the 18S gene. In order to evaluate the extent of DNA damage, we selected two fragments of mtDNA (79bp and 230bp in length) as target templates for a qPCR array using the 18S gene as an internal reference. The extent of mtDNA damage was parallel to the value of 79bp/230bp. The primers used for the real‐time PCR are listed in Table 2.11The Mitochondria and Nuclear Extraction Kit for Cells (Beyotime Biotechnology) was used to isolate nuclear and mitochondrial fractions from each experimental sample. In each case, we followed the manufacturer's guidelines.2.12A plasmid that overexpressed TP53, along with a negative control (NC) plasmid, were constructed by GenePharma Co., Ltd. Cells were transfected with the plasmids using the Thermo Scientific™ TurboFect™ Transfection Reagent in accordance with the manufacturer's protocol. In brief, cells were plated in 6‐well plates and transfected the next day with 4 μg of plasmid using 6 μl of transfection reagent. Cells were harvested 24 h after transfection, and whole‐cell lysates were isolated for Western blotting. The sequence of TP53 is as follows: ATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGA.2.13Tumour tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Paraffin sections were cut into sections (4 μm thick) and then deparaffinized and rehydrated prior to antigen retrieval. Sections were blocked with 10% bovine serum albumin in TBS‐Tween 20 (Sigma‐Aldrich) for 1 h at room temperature. Sections were then incubated overnight at 4℃ with primary antibodies against P53 (21891–1‐AP) and PINK1 (23274–1‐AP). The following morning, sections were incubated with secondary antibodies for 30 min at 37℃. Then, slides were incubated with diaminobenzidine for 5 min, and then counterstained with Gill's haematoxylin for 30 s. Images were captured on a microscope (Discover ECHO Company).2.14Tumour tissues from the different treatment groups were fixed in 2.5% glutaraldehyde and then embedded in an embedding agent. Next, specimens were sliced into sections (50–70 nm in thickness). The sections were then dyed with uranyl acetate and citrate dye solution. The TEM system (Thermo Fisher Scientific Company) operated at an acceleration voltage of 80 kV and an electron tomography voltage of 120 kV. Specimens were magnified by ×150,000, and images were acquired. Three tumour specimens in each treatment group were randomly selected for TEM examination. And each specimen outputted a clear picture of which the mean length of mitochondria was measured directly by the software of TEM. Then, the data were analysed.2.15® easyCyte flow cytometer (Merck KGaA).Apoptosis assays were performed using an Apoptosis Detection Kit (BD Biosciences). Cells from different treatment groups were trypsinized with 0.25% trypsin, and assays were conducted in accordance with the manufacturer's protocol. In total, 10000 cells were counted in each treatment group using a Guava® easyCyte flow cytometer (Merck KGaA).For the analysis of mitochondrial mass, cells were grown in 6‐well plates and treated with the different agents. Cells were trypsinized with 0.25% trypsin and suspended cells were stained with MitoTracker™ Red FM (Invitrogen) (1:10000) for 30 min at 37℃. Cells were then rinsed three times with PBS, and 10,000 cells were counted for each treatment group using a Guava® easyCyte flow cytometer (Merck KGaA).The production of ROS was measured by a ROS Assay Kit (Beyotime Biotechnology). In brief, cells were collected and resuspended in DMEM medium after treatment and then incubated with DCFH‐DA for 30 min at 37°C. Cells were then rinsed three times in PBS, and 10,000 cells were for each treatment group using a Guava2.16U87 cells were seeded at a density of 8000 cells/well and treated for 24 h with the respective treatments . On the day of the assay, cell culture medium was removed and replaced by assay medium supplemented with agents according to the protocol and the Seahorse XFe96 analyzer was used. In the test, basal cellular oxygen consumption was determined, followed by injection of the ATP synthase inhibitor oligomycin (5 µM), the uncoupler fluoro‐carbonyl cyanide phenylhydrazone (FCCP) (2 µM) and the complex I inhibitor rotenone (0.5 µM) combined with the complex III inhibitor antimycin A (0.5 µM). The oxygen consumption rate was detected, and each measurement was normalized to cell content.2.17t test. Analysis of variance (ANOVA) was also used to conduct multiple comparisons using GraphPad Prism 7.00 . Dunnett's post hoc multiple comparisons test was used after ANOVA. Data are presented as means ± standard deviation, and p < 0.05 was considered to indicate a statistically significant difference.Data from three independent experiments were collected and analysed using SPSS 22.0 (IBM Corp.) and the Student's 33.1As an alkylating agent, TMZ can induce damage to nuclear DNA and mitochondrial DNA.In order to identify the mechanisms underlying the increased level of oxidative phosphorylation, we first detected the mass of mitochondria in the two cell lines, using three methods: (1) labelling mitochondria with specific fluorescent probes Figure A, 2) by by2B) an3.2The mass of mitochondria within a cell is precisely controlled by mitophagy.3.3In order to further explore the mechanism by which mitophagy was downregulated, we focused on how the expression of PINK1 was regulated. We also investigated the elevated expression of intracellular nuclear transcription factor (NTF) in tumour cells under TMZ treatment. First, we found that TMZ treatment‐induced cellular DNA damage responses in both SHG44 and U87 cells; these responses involved high expression of γ‐H2A.X and the increased aggregation of the protein in the nucleus  establishing a cell model overexpressing TP53 by plasmid transfection; under this condition, we observed that PINK1 expression was downregulated in cells overexpressing TP53, and that the expression of Ser65 phosphorylated ubiquitin was correspondingly downregulated  after TMZ treatment, the tumour cells showed reduced levels of ATP; thereafter, however, the levels of ATP increased. The curve created by plotting ATP concentration with time showed two troughs; one at 10 min and the other at 6 h Figure A. FurtheWhat is more, to confirm that the AMPK regulated the function of mitochondria, we performed the mito stress test by using the seahorse analyzer and revealed that: 1.TMZ elevated the OXPHOS potential of mitochondria while repressing the activity of AMPK impaired the effect Figure D.3.6In order to verify that the mobilization of mitochondrial dynamics was involved in the metabolic reprogramming of tumour cells under chemotherapy stress and to explore whether interfering with mitochondrial dynamics could sensitize GBM to TMZ treatment, we used two different agents: WY14643 to promote mitochondrial fissionFinally, we found that WY14643 promoted mitochondrial fission and increased the efficacy of TMZ chemotherapy. This treatment also enhanced the efficacy of TMZ to induce apoptosis of SHG44 and U87 cells . Renxuan Huang: Software ; Visualization . Kunmeng Yang: Data curation . Yichun He: Writing – review & editing . Delu Dong: Supervision . Yufei Gao: Conceptualization (lead); Funding acquisition (lead).Fig S1Click here for additional data file.


Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of  •Vein-specific enhancers can contain essential ETS motifs.•VEGFA induced an increase in ETS binding at vein, arterial and angiogenic enhancers.•VEGFA stimulation cannot induce vein-specific enhancer activity. The vascular system is subdivided into arteries, veins, lymphatics and capillaries, each comprised of genetically distinct ECs expressing specific fate-determining genes . The essE-26 transformation-specific) transcription factor family  identified a group of ETS binding motifs required for arterial and angiogenic activity  signalling may act via ETS transcription factors to enable specific activation of arterial and angiogenic genes. VEGFA signalling influences many processes during early vascular growth, and plays essential roles in vasculogenesis, arterial specification and angiogenesis . ETS traactivity . ERG binactivity . A similactivity , whilst activity .Despite their hypothesised role in arterial and angiogenic-specific patterns of gene expression, binding motifs for ETS factors are also a common feature of many pan-endothelial expressed gene promoters and enhancers . ETS1, Ein vivo. These results indicate that within the endothelium, VEGFA-stimulated ETS factor binding is a shared feature at enhancers associated with multiple different patterns of gene expression, and suggests that additional transcription factors may be primarily responsible for directing arterial, angiogenic and venous-specific gene expression patterns downstream of different growth factor signalling inputs.In this paper, we undertake a detailed analysis of two recently characterized vein-enriched gene enhancers. We demonstrate that, similar to arterial and angiogenic enhancers, ETS factor binding at these venous enhancers is necessary for enhancer activation and vein-specific patterns of reporter gene expression, and that this binding is also enriched by VEGFA signalling. However, unlike arterial and angiogenic enhancers, these venous enhancers cannot be directly activated by over-expression of VEGFA 22.1Ephb4 and Coup-TFII (Nr2f2) gene loci (Coup-TFII. Neither enhancer was active in the mature microvasculature (We have recently identified enhancers within the venous-enriched ene loci . Both enA/T)  were able to compete for binding of either ETS1-DBD or ERG, or both proteins   and Fig.A/T) A. Of theCGGAAG/A . We perfCGGAAG/A B–C and fCGGAAG/A D–E, bothproteins C while Eproteins E. The spproteins C and E. EPHB4 and COUP-TFII/NR2F2 were significantly enriched in these HUVECs . Similar to the venous enhancers investigated, ERG binding around these arterial-, angiogenic- and pan-endothelial-expressed enhancers was seen in both HUVEC and HUAEC cells, suggesting that binding of ERG to specific enhancer regions was not routinely restricted to the EC subtypes in which the associated genes are preferentially active . Taken together, these results demonstrate that functional ETS binding motifs can be present within vein EC-specific enhancers, and show that the ability to bind ERG and other ETS factors is not restricted to enhancers that are active in arterial and angiogenic ECs.Since the binding of ETS factors to vascular enhancers has been previously associated with arterial-specific and angiogenic-specific enhancers, we next investigate whether ETS factor binding was also a feature at venous-specific enhancers . We found significant binding for ERG, FLI1 and ETS1 at both Ephb4-2 and CoupTFII-965 venous enhancers . EMSA analysis confirmed that the mutated ETS binding motifs could not bind ETS proteins   exclusively to the venous endothelium .In agreement with previous  studies , our resproteins C and E. on (hpf)  and Fig.on (hpf) A–B. Howeon (hpf) A–B. We sothelium  and Fig.othelium C–D, the The loss of Ephb4-2 and CoupTFII-965 vein enhancer activity after ETS motif mutation observed here is similar to the loss of activity seen when ETS motifs are mutated in the arterial and angiogenic Dll4in3 enhancer , in othe2.3erg and fli1. There are multiple ETS factors expressed in the developing zebrafish vasculature  zebrafish was significantly reduced after morpholino knockdown of erg and fli1, with the strength of Ephb4-2:GFP transgene activity inversely correlated with the levels of erg/fli1 MO . Conversely, we saw a reduction in the expression of endogenous ephb4 and stab1l, a zebrafish venous marker, when assessed by whole mount in situ hybridization analysis . Similar reductions in the expression of some venous-enriched genes was also observed after ERG depletion in HUVECs  transgenic zebrafish, although this was not as marked . As previously reported, erg/fli1 knockdown also resulted in reduced activity of the arterial/angiogenic Dll4in3 enhancer in transgenic zebrafish  and tg(CoupTFII-965:GFP) zebrafish lines compared to the arterial (tg(Dll4in3:GFP) zebrafish line. At the lowest concentration of inhibitor, Dll4in3:GFP activity was more notably reduced than either venous enhancer, while higher SU5416 doses significantly reduced activity of all enhancers  results in EC apoptosis, lower levels of VEGF inhibition result in reduced arterial and venous marker gene expression, although the reduction in venous genes was less pronounced and sometimes compensated by expansion of vein gene activity into the dorsal aorta . We therAlthough Dll4in3:GFP was more sensitive to inhibition of VEGFA signalling than either the venous Ephb4-2:GFP or the CoupTFII-965:GFP, our previous results show that changes in ETS factor binding after VEGFA stimulation can be seen at both venous and arterial enhancers. Consequently, it is unlikely that changes in ETS factor occupancy at enhancers can explain the differences between venous and arterial enhancer responses to VEGFA inhibition. An alternative explanation may be that additional transcription factors  may be instead responsible for allowing arterial and angiogenic enhancers a greater sensitivity to VEGFA signalling.2.6in vivo. We first used an established model of VEGFA-stimulated blood vessel growth in mice, in which an adenovirus expressing VEGFA164 (Ad-VEGFA164) is injected intradermally into the ears of adult mice . In both enhancers, angiogenic expression was lost by 60 days after injection, a time-point known to be independent of VEGFA signalling.We next examined whether VEGFA over-expression, and subsequent increased ERG occupancy, is alone sufficient to initiate activity of venous-, arterial- and angiogenic-specific enhancers ult mice . This reult mice . Vessel ult mice . To deteenic ECs A. Ad-VEG164 injection can specifically re-activate the Dll4in3 and HLX-3 enhancers in their native EC sub-types. However, they cannot determine whether this occurs via a VEGFA-mediated increase in ERG binding, VEGFA-mediated increase in other transcription factors binding to these enhancers or VEGFA-mediated removal of repressive factor binding. If VEGFA-mediated activation of these enhancers occurs primarily via changes to ETS factors, we would expect a similar reactivation of venous enhancers. We therefore next determined if Ad-VEGFA164 injection was able to directly activate the Ephb4-2:lacZ and CoupTFII-965:lacZ transgenes. Unlike with the arterial and angiogenic enhancers, we observed no endothelial transgene activity at any time point in either Ephb4-2:lacZ or the CoupTFII-965:lacZ Ad-VEGFA164 injected ears, although occasional ectopic expression could be detected  embryos (following protocol and concentration from tg(Ephb4-2:GFP) embryos . This did not change significantly when we increased the amount of vegfaa injected . This result therefore further indicates that increased VEGFA signalling does not increase Ephb4-2 enhancer activity. Additionally, at 50 pg vegfaa121 and vegfaa165 we saw no clear expansion of Ephb4-2:GFP expression beyond the venous endothelial expression pattern observed in control embryos (vegfaa121 and vegfaa165 mRNA into 1-cell stage tg(Dll4in3:GFP) embryos resulted in slightly increased GFP intensity and expansion into the caudal vein plexus, as previously described by vegfaa levels . Taken together, these results suggest that the failure of Ad-VEGFA164 to activate venous gene enhancers in the mouse ear is unlikely to be a result simply of developmental context. Furthermore, the Ephb4-2:lacZ transgenes can be reactivated in injured neonatal hearts , suggesting that absence of normal activity does not affect enhancer reactivation. Taken together, these results indicate that VEGFA stimulation is not sufficient to activate transcription from the Ephb4-2 venous EC-specific enhancer, despite its reliance on VEGFA-augmented ETS transcription factors.Although developmental arterial and angiogenic enhancers were reactivated in the adult mouse ear by Ad-VEGFA164 A and B,  reduced . Furthern plexus . To dete3The role of ETS transcription factors in the regulatory hierarchy of endothelial cells has been unclear. The specification and maintenance of the endothelial cell lineage requires the shared activation and repression of many lineage-defining genes. However, differential gene expression within specific sub-populations of endothelial cells is also essential for vascular function. Consequently, spatial and temporal control of endothelial gene expression must involve multiple layers of regulation. While analysis of arterial and angiogenic-specific enhancers has supported a proposal that ETS factors play a specific role in the activation of arterial and angiogenic genes downstream of VEGFA signalling, ETS factors have also been implicated in the more general activation of genes, and their cognate regulatory elements, involved in endothelial identity and maintenance. However, the analysis of the precise roles played by ETS factors in the vasculature has been complicated by the abundance of different ETS factors in the endothelium coupled with extensive redundancy between different ETS family members. Parsing their two potential functions is further challenged by the multiple roles played by VEGFA signalling in the vasculature. Recent analysis in zebrafish concluded that low levels of VEGFA signalling promotes general endothelial identity and survival, while higher levels of VEGF signalling primarily promotes arterial specification . ConsequIn this paper, we have clearly demonstrated that vein-specific gene enhancers can be reliant on ETS factors for activation in venous ECs. This is similar to that previously reported in arterial-specific and angiogenic-specific enhancers, even though expression of venous genes is not induced to the same extent by high VEGFA signalling. Further, we have shown that VEGFA signalling can also increases ETS factor binding at venous enhancers, indicating that selective arterial and angiogenic gene activation is unlikely to be achieved through this mechanism. Because our analysis was predominantly restricted to two venous enhancers, the conclusions may not equally apply to all venous-expressed genes. Of note, Notch1 expression in arterial ECs , Tg(CoupTFII-965:lacZ) Tg(Dll4in3:lacZ) and Tg(HLX-3:lacZ) were generated as previously described  containing 5 mM potassium ferrocyanide, 5 mM ferricyanide, 0.1% sodium deoxycholate, 0.2% Nonidet P-40, 2 mM MgCl2 and 1 X PBS. After staining, embryos were rinsed through a series of 1 X PBS washes, then fixed overnight in 4% paraformaldehyde at 4 °C. All embryos were imaged using a Leica M165C stereo microscope equipped with a ProGres CF Scan camera and CapturePro software (Jenoptik). In instances that images have been altered to improve quality and colour balance, each image within a set have been altered using the same parameters. This occasionally included to selective depletion of the yellow or red colour channel, in order to counteract issues from the X-gal stain solution (which is orange). All samples are stored in 4% PFA indefinitely and slowly become less orange. Consequently, samples stained more recently have a greater yellow/orange hue. An example of this alteration can be seen in  and tg(CoupTFII-965:GFP) zebrafish lines were generated in  at 28.5 °C. To image, all embryos were dechorionated and anesthetized with 0.01% tricaine mesylate. For analysis of F0 transgenic zebrafish, single embryos were transferred into a flat bottom 96-well plate, and GFP reporter gene expression screened with a Zeiss LSM 710 confocal microscope at 46–50 hpf. Whole zebrafish were imaged using the tile scan command, combined with Z-stack collection under a confocal microscope Zeiss LSM 710 MP (Carl Zeiss) at 488 nm excitation and 509 nm emission (EGFP).All animal procedures comply with all relevant ethical regulations, were approved by Clinical Medicine Local Ethical Review Committee, University of Oxford and licensed by the UK Home Office. Stable rated in . F0 mosarated in . EmbryosFor pharmacological inhibition of VEGF signalling, embryos were manually dechorionated and 0.625 μM, 1.25 μM, 2.5 μM and 5 μM of SU5416 (Stratech Scientific Ltd.) added at approximately 5ss as described in . Control4.3Ephb4-2mutETS and CoupTFIImutETS enhancer sequences were initially generated as custom-made, double-stranded linear DNA fragments . DNA fragments were cloned into the pCR8 vector using the pCR8/GW/TOPO TA Cloning Kit  following manufacturer’s instructions. Once cloning was confirmed, each enhancer was transferred from the pCR8/GW/enhancer entry vector to a suitable destination vector using Gateway LR Clonase II Enzyme mix  following manufacturer’s instructions. For mouse transgenesis, the enhancer was cloned into the hsp68-LacZ-Gateway vector (provided by N. Ahituv). For zebrafish transgenesis, the enhancer was cloned into the E1b-GFP-Tol2 vector (provided by N. Ahituv).4.3.1AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTTTCCTGAGGCTGTTTCCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTTCCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTTCCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTTCCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGGAAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.4.3.2agCTGAGGCTGTTagCTGTCTGGCTCCTGGGGGCCCTCGGGATGGCTGGGAGGGCCCTagCTCTCATTTGCTAGCACCCCCTCTCATCCATCAGTTTGAGGGGAGGGTCCAGGAAAGACGGCCTCCTATCTACATCAGGGCACTGTGAGTGTGGGGCACGGGATGGTTGGATGAGAGAGGTGCTGTTCCCGAAGTCGGTCCTTTAAGGGCTGCGGTAAGGAGACTTTAATTTAAGGTAATTAGTACAGGGTCTGGAAACTCTGAGGTAGGAGTCTGGGGCACCTGGGAGTCTGCCAAATACCCTAAGGGCGCACACACACACCCCAGCGGGCGACCGGTGATGACCTCTTGTCCGCCTGCGCGCACACACACACCAGCGGGCGCGGGAGACCCGTGATGGCCTTTTGTCCCCGTGCACTTATCTagCTGGCGCAAGTAGTGCTCCCCACCCCCTGCCCTagCTCACAGCCCTGCCTGGGTCCCGCTCCGGGGTGGGTCAGCCAGGGCAGAGAACAGCCGGCTTGGCTGGAGCCAGGCTGACCGGCTAGATCTGGGAGTCCCCTCCTCCTTCCCCACGCAGACTCAGGCTCCCCTTCTCTTATCCACAGACACCCCCTTTTTTGCAGCTATCATTCTGCATCCGGGTCCCCCTGAATTTCTGAGTCGTGGCTTGTTCTCAC.AATCAGTGCGTGCTCGTTAAGTCCTGGAGATCCACTGAGCGCGCAGCCTAACGCTGGAGAAAGTGGTTTGAAACCCAAAGTATAGAAAATGTAAATAAAAGGCAGGCGTGTCAGAGAGGGTGAGGGATCTCCGTAACACCTCATTTCATTTTTTTAAAGGAGGGGGACACTTCCCCGCCGCCTGCAGCCTTGACCTCCAAGGCGGGGGTAGGGACCGTTGTGGCTCTT4.3.3GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAGGAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACATCCTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAGGAAATGATCGAGATGCCTTGTCAGATCTTC.4.3.4GCTGAGACAAATGGAAAGCTGAAGATAAGGATCCTCTGAGGTGCGAACATACAGCTGTTGGGAATTGCCAGAGAATCGGACCAATAAAtcAAGTCACTATTTTTCCAGGCCTGAAGTGAGTTATAGGGCGAGACGGGTGTTGTATATTTATGTAAGGCAACAGCAGGGAGTTTAAGCGGCTGGATATTGCTGAAAGAGCATCATTCACATTCAGGCGGAGACAAAAGGTGGAAATGAAGCAACagCTTGGCCAAAGAAGGCCTCAAGACAGAATAATAACAGTTCAGAGAGGGGGGCTGTGTGCACGGCCGAGGGTCGGCCTCAAAACCAtcAAATGATCGAGATGCCTTGTCAGATCTTC.4.4Mouse and human sequences of putative enhancers were aligned using ClustalW . Binding4.5italic underlined nucleotides modified (GGA to TCA or TCC to TGA) in mutant version:Proteins were made using the TNT Quick Coupled Transcription/Translation system as described in the manufacturer’s directions. The truncated ETS1 DNA binding domain (ETS-DBD) and full length ERG were in the pCITE2 plasmid, and transcribed using T7 polymerase. To make each probe, double stranded oligonucleotides with CTAG 5′ overhangs were labelled with 32P-dCTP using Klenow (Promega) to fill in overhanging 5′ ends, and purified on a non-denaturing polyacrylamide-TBE gel. 20 μl binding reactions consisted of 2–3 μl protein or lysate control, 2 μl 10X binding buffer  and 0.5 μg poly dI-dC. For competitor lanes, a 100-fold excess of competitor DNA was added in a volume of 1 μl. Binding reactions were incubated at room temperature for 10 min before the addition of radiolabelled probe, after which they were incubated an additional 20–30 min. Gels were electrophoresed on a 10% non-denaturing polyacrylamide gel. Sequences of the probes and competitor regions are listed below, with tccccgccg;ETS control probe  CTAGtaaatcctgaggctg.Ephb4-2 ETS-b CTAGgttgtggctctttcctgtctggc.Ephb4-2 ETS-c CTAGgaggctgttggatggctggga.Ephb4-2 ETS-d CTAGggccctcgtcctctcatttg.Ephb4-2 ETS-e CTAGagggccctggatggttgg.Ephb4-2 ETS-f CTAGgtggggcacgggatgagagaggtgc.Ephb4-2 ETS-g CTAGgatggtttcctggcgcaagta.Ephb4-2 ETS-h CTAGcacttatcttcctcacagcccEphb4-2 ETS-I CTAGcctgccctggaaacagcc.Ephb4-2 ETS-j CTAGagccagggcaggaaagctgaagataa.Coup-965 ETS-a CTAGacaaatggatcctctgag.Coup-965 ETS-b CTAGgctgaagataaggaattgccagaga.Coup-965 ETS-c CTAGagctgttgggaagtcactat.Coup-965 ETS-d CTAGcggaccaataaaggaaatgaagcaacatc.Coup-965 ETS-e CTAGaaggttcctggccaaag.Coup-965 ETS-f CTAGaagcaacaggaaatgatcgagatc.Coup-965 ETS-g CTAGcaaaaccatccaggcctg.Coup-965 ETS-h CTAGgtcactatttt4.6165 (PeproTech) at 25 ng/ml for 1.5 h. Cells were then trypsinised and the cell pellet collected.For VEGF stimulation experiments in cells, human umbilical vein pooled endothelial cells  were grown in Endothelial Cell Growth Medium 2 with BulletKit (PromoCell). Media was changed every 48 h. Four 80% confluent 15 cm dishes per condition were serum starved in 0.5% Foetal Bovine Serum (Gibco) overnight before being stimulated with VEGFAChromatin immunoprecipitation was carried out as previously described . BrieflyImmunoprecipitated DNA was analyzed by qPCR using TaqMan Custom Gene Expression Assay Probes (ThermoFischer) designed against 100bp regions of the Ephb4-2 enhancer, the CoupTFII-965 enhancer or a gene dessert region of Chromosome 7 as a negative control.4.6.1ACCCCTGCCCTTCCTTGCTGTTCTGCCTGGGTCCTGCGCCCGGGTTGGGGGGGGTGGGCCGGTCACCGAGGGCAGGAAACAGCCGGCTTCACTGGAGCCAGGCAGACCAG.4.6.2AGCGGCTGTATATTGCTGAAAGAGCATCATTCACATTCAGGCAGAGACAAAAGGTGGAAATGAAGTAACATCCTGGCTGAAGAAGGCCTCACGACAGAATA.4.6.3CCTCAGCCTCCCAAGTAGCTGGGATTACAGGTGTGTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGAACTCAGG TGATCTA.Each ChIP was performed on at least three biological replicates, with three technical replicates for each. Statistical analysis was performed in StepOne plus software, Microsoft Excel. Input was taken as the supernatant from the non-antibody control condition. Results are expressed as the mean of the % input defined as 100∗(2^(adjusted Input ct – ct IP)) across all replicates. Significant differences were calculated using ANOVA f test with p values subsequently derived from Tukey HSD test, accounting for multiple comparison correction. Graphs were produced using R statistical package.4.7https://ncbi.nlm.nih.gov/geo/) under accession GSE93030. The four ChIP-seq datasets used have accession numbers GSM2442775 to GSM2442778. Data consisted of ChIP-seq Model-based Analysis of ChIP-Seq (MACS) (https://CRAN.R-project.org/package = data.table and https://CRAN.R-project.org/package=ggpubr). GRCh37 ROIs: HLX-3 chr1:221,049,659–221,050,776, Dll4in3 chr15:41,222,807–41,223,778, Ephb4-2 chr7:100,426,194–100,427,393, and CoupTFII-965 chr15:95,908,708–95,909,301.ChIP-seq analysis was conducted on the published and publicly available data from q (MACS)  regions q (MACS) . MACS pe4.8Antisense MOs were ordered from GebeTools LLC and dissolved in water before injected into 1–2 cell stage zebrafish embryos as previously described . Sequencfli1 MO (3 – 6 ng) TTTCCGCAATTTTCAGTGGAGCCCG (GGAGCCCG .erg MO (3 – 6 ng) CAGACGCCGTCATCTGCACGCTCAG .4.9in situ hybridization ephb4, efnb2 and stab1l probes were generated as custom-made, double-stranded linear DNA fragments , cloned into the pCR2 vector using the TOPO/TA cloning kit (Invitrogen 450641) and transcribed using SP6 and T7. The sequences are provided below. dll4 probe was kindly provided by R. Patient, University of Oxford, Oxford. Whole-mount in situ hybridization was conducted as previously described . Embryos were washed through a dilution series of 2 x SSC followed by 0.2 x SSC at 65 °C and thereafter taken through room temperature dilution washes of 100% MABT . Nonspecific sites were blocked with MAB block (MABT with 2% Boehringer block reagent) and the embryos incubated for 15 h with 1:4000 antiDIG antibody (Roche) at 4 °C, before washing in MABT. Prior to staining, embryos were washed in AP buffer and the in situ signal developed at room temperature with BM Purple (Sigma-Aldrich). Staining was stopped as appropriate, and embryos were bleached in 3% H2O2/0.5% KOH until pigmentation disappeared, then re-fixed in 4% PFA for 20 min and washed 4 times with PBST. Embryos were transferred to 80% glycerol for imaging.For zebrafish whole-mount escribed . Brieflyin situ/condition. Experimental blinding was not used as phenotypes of control and treated were easily detectable due to morphological defects.Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern. Numbers of zebrafish embryos was no less than 30/4.9.1TCTCAGCTCTGGACAAGCTGATCCGCAACCCGGCCTCACTCAAAATCACAGCGCAGGAGGGGGCGGGCCCCTCTCACCCTCTGCTGGACCAGCGGTCTCCACTCACGCCCTCATCCTGCGGGACAGTGGGTGACTGGCTGCGGGCCATCAAGATGGAGCGCTACGAGGAGACATTTCTGCAGGCGGGATACACGTCCATGCAGCTCGTCACCCACATCAACACGGAGGATCTGCTGCGTTTGGGAATAACTTTAGCAGGTCACCAGAAGAAGATTCTCTCCAGCATTGAGGCTCTCGGGATTCAAAACAAAGCACCAGGGAATGTGCTGTACTGA.4.9.2AAAACCAAGTCGATGAAAATCATCATGAAGGTTGGACAAAACCCCTCTGATCCCATTTCCCCCAAAGACTACCCTACCAGTTACCCTCCCAAACACCCTGACTTAGGGGGCAAGGACAGCAAATCGAATGAAGTACTTAAGCCAGATGCATCTCCTCATGGGGAAGATAAGGGAGATGGAAATAAATCCTCATCAGTCATTGGCTCAGAGGTGGCCCTGTTTGCCTGCATCGCCTCAGCAAGCGTCATCGTCATCATCATAATCATCATGCTAGTTTTCCTTCTCCTGAAGTATCGACGA.4.9.3GGATTCAGCAGCTACAGACACACCCAACCTCATCGACTAGCACAGACAGCAGCGTTAAACTCTCCCTTCATTTACCTGCAATCAGCTGACCGCTCTTAAAATAAAGGTTCTGTATTGGCATTGATGGTTCCGCGAAGAATCTTTATAAGCCATAACATCTTTCCATTTCCATGAGGTGTAAAAAGACTCTTTAGAATATTAAAATGTTACTTCATAAACATTTGATGTGTTTGATTGCAGATACTTCAGAGTGTTTAACTTCCACCCATTTATTTCTGCGTTTCACACATATTTTTGACTAAAAATGTTCTTTACATTAAGAAAAAATGGTGTACTCACCCTCAAGTAGGTCCAAACCTTCACGAGTTTCTTTTTTTGCCTTCTGTTGAACACAAACAAGAAAATATTTTGATAATGCGTTAAGCAAGGGGCCATTAGCTGTTTTGATCCAACTTTTTTCATGCGCATTTTAAATTATCGCATGTAAAAAAGCTTAATGGAAACCCAAGATGTGCTTAATTTGTCAAAATCTGCAT.4.10164 injections were performed on nude mice as described in (164 (provided by Lilly) diluted 1:30 in sterile 3% glycerol/PBS. At the required time-point after injection, ears were harvested and skin removed from the dorsal side. Ears were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 20 min at 4 °C, washed twice in PBS then X-gal stained overnight at room temperature. Ears were then placed in 4% paraformaldehyde for storage.Intradermal Ad-VEGFAribed in . Briefly164 injected ears, ears were harvested and X-gal stained as described above, then dehydrated through a series of ethanol washes, cleared by xylene and paraffin wax-embedded. 5 or 6-μm sections were prepared and de-waxed. For imaging of X-gal staining, slides were counterstained with nuclear fast red (Electron Microscopy Sciences).For histological analysis of Ad-VEGFA4.11vegfaa121 and pCS2+vegfaa165 plasmids were kindly provided by S. Sumanas, Cincinnati Children’s Hospital Medical Center, Ohio, USA. vegfaa121 and vegfaa165 mRNA was synthesised in vitro using the mMessage mMachine SP6 transcription kit (ThermoFisher Scientific) and injected into 1 cell stage zebrafish embryos at a final concentration of 50 pg of mRNA per embryo. Analysis was qualitative not quantitative, therefore no statistical analysis was applied to the observations of staining intensity and pattern.pCS2+No competing interests declared.


The rat progression elevated gene-3 (PEG-3) promoter displays cancer-selective expression, whereas the rat growth arrest and DNA damage inducible gene-34 (GADD34) promoter lacks cancer specificity. PEG-3 and GADD34 minimal promoters display strong sequence homology except for two single point mutations. Since mutations are prevalent in many gene promoters resulting in significant alterations in promoter specificity and activity, we have explored the relevance of these two nucleotide alterations in determining cancer-selective gene expression. We demonstrate that these two point mutations are required to transform a non-cancer-specific promoter (pGADD) into a cancer-selective promoter (pGAPE). Additionally, we found GATA2 transcription factor binding sites in the GAPE-Prom, which regulates pGAPE activity selectively in cancer cells. This newly created pGAPE has all the necessary elements making it an appropriate genetic tool to noninvasively deliver imaging agents to follow tumor growth and progression to metastasis and for generating conditionally replicating adenoviruses that can express and deliver their payload exclusively in cancer.Progression-elevated gene-3 (PEG-3) and rat growth arrest and DNA damage-inducible gene-34 (GADD34) display significant sequence homology with regulation predominantly transcriptional. The rat full-length (FL) and minimal (min) PEG-3 promoter display cancer-selective expression in rodent and human tumors, allowing for cancer-directed regulation of transgenes, viral replication and in vivo imaging of tumors and metastases in animals, whereas the FL- and min-GADD34-Prom lack cancer specificity. Min-PEG-Prom and min-GADD34-Prom have identical sequences except for two single-point mutation differences (at −260 bp and +159 bp). Engineering double mutations in the min-GADD34-Prom produce the GAPE-Prom. Changing one base pair (+159) or both point mutations in the min-GADD34-Prom, but not the FL-GADD34-Prom, results in cancer-selective transgene expression in diverse cancer cells  vs. normal counterparts. Additionally, we identified a GATA2 transcription factor binding site, promoting cancer specificity when both min-PEG-Prom mutations are present in the GAPE-Prom. Taken together, introducing specific point mutations in a rat min-GADD34-Prom converts this non-cancer-specific promoter into a cancer-selective promoter, and the addition of GATA2 with existing AP1 and PEA3 transcription factors enhances further cancer-selective activity of the GAPE-Prom. The GAPE-Prom provides a genetic tool to specifically regulate transgene expression in cancer cells. Defining the steps necessary to convert a normal cell into a cancerous one has been aided by identification and interrogation of defined genetic elements that regulate these processes ,2,3. Usipeg-3 and gadd34 genes share 73% nucleotide and 59% amino acid sequence homology [peg-3 and the murine gadd34 genes [The N-terminal domain first 415 aa) of rat PEG-3 is identical to the rat GADD34 protein  aa of ra,8. The p34 genes ,11,12,1334 genes . In cont34 genes . These speg-3 and gadd34 genes is chiefly transcriptional [Previous studies indicate that regulation of both the iptional . The fuliptional ,16. Promiptional ,15,16. Siptional . Furtheriptional ,15,16.Mutations are common events in gene promoters, which can result in dramatic changes in promoter specificity and activity ,19,20,21To determine why the cancer-specific min-PEG-Prom (pPEG) is functionally different than the pGADD, which shares almost identical sequence similarity, we compared the sequence of the promoter regions of the pPEG  with the pGADD , which resulted in the identification of only two base pairs that were different . Using sUsing the luciferase assay, we compared the activity of the pGADD-, pPEG- and pGAPE-Luc in cancer vs. normal  human cells. As shown in Previous studies have confirmed that when pPEG-Luc or pPEG-HSV-Tk were complexed with in vivo jetPEI a linear polyethylenimine (l-PEI), intravenous injection permitted imaging of both primary tumors and metastases . We initWe used in vivo jetPEI for delivery and imaging of the different promoters ,27. JetPAn important question was whether a single mutation in the pGADD was sufficient, or if both mutations were necessary, for pGAPE to display cancer-specific activity. To accomplish this goal, we generated a pGADD with a single mutation at bp −260  and a second pGADD mutation at bp +159 . Using the same in vitro Luc-assay-based approach (described above), we analyzed the activity of pGADD, pPEG, pGAPE, pGADD1-1 and pGADD2-2 in the same series of human cancer, human immortalized normal cell lines, and primary normal cells shown in pPEG and pGAPE show tumor-specific in vivo imaging capabilities as well as enhanced tumor-specific expression in vitro, whereas the full-length GADD promoter, pT-GADD, does not show tumor specificity compared to normal cells. The same is true with the single mutation modification of bp −260 (pGADD1-1), which lacks cancer specificity, whereas the change of bp +159 (pGADD2-2), shows cancer selectivity when engineered in pGADD (the minimum GADD34-Prom). To check the transcription factors involved in this cancer-selective activity, we analyzed the pGADD with and without mutations with the TFBIND software , which pChromatin immunoprecipitation (ChIP) assays confirmed an interaction between GATA2 and pGAPE in DU-145 and RWPE-1 cells A. DU-145We next determined whether changing GATA2 expression could modulate promoter binding to GATA2. RWPE-1 and DU-145 were transfected with GATA2 OE plasmid or shGATA-2 (inhibitory) plasmid for 48 h and then processed for ChIP assays. We predicted and confirmed that, following overexpression of GATA2, the binding to pGAPE/pPEG increased as compared to non-transfected cells, and conversely, GATA2 inhibition reduced binding to the pGAPE/pPEG. However, binding to the pGADD was not altered in either of these cell lines under these conditions C. CollecTo determine if a single mutation or both mutations (bp −260 or bp +159) in the full-length GADD34-Prom  could confer cancer selectivity, we developed total pT-GADD constructs with a single mutation at G-A, pT-GADD1-1-Luc or C-T, pT-GADD2-2-Luc, and with both the mutations, pT-GADD2-Luc . The CTV induces profound anticancer activity in vitro and in vivo in diverse cancer indications [Tissue- and cancer-selective/specific promoters also provide functional tools to potentially regulate virus replication in a conditional manner, including adenovirus, herpesvirus, human immunodeficiency virus, simian immunodeficiency virus, and others ,32,59,60-7/IL-24 , under cications ,30,31,32In summary, we document that two-point mutations are critical to convert a non-cancer-specific pGADD into a cancer-selective pGAPE. A single mutational change at bp +159 of pGADD to that of the pPEG also promotes cancer selectivity both in vitro and in vivo. Additionally, we define a new transcription factor, GATA2, to which binding to the GAPE-Prom is indispensable for cancer-selective expression and activity. This newly engineered pGAPE embodies all the necessary features that make it an ideal tool to regulate transgene expression in a cancer-selective manner to both deliver imaging agents noninvasively to follow tumor promotion and metastasis and create CRADs expressing replicative functions uniquely in cancers.2 at 37 °C. Cells were validated for authenticity in prior studies [Immortalized human prostate epithelial (RWPE-1) and prostate cancer  cells, human immortalized pancreatic mesenchymal (LT-2) and pancreatic cancer  cells, early passage primary human mammary epithelial (HMEC) and breast cancer (MDA-MB-231 and SUM159) cells, H-TERT immortalized primary human fetal astrocytes (IM-PHFA) and neuroblastoma  cells were cultured as described previously ,64,65. S studies ,64,65 an studies ,64.® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) was purchased from Cell signaling technology. Dual Luciferase Reporter Assay Kit was obtained from Promega  and used in the study. Lipofectamine 2000  and Invivo JetPEI  and transfection reagents, D-Luciferin Potassium Salt Bioluminescent Substrate , Agilent Quickchange II site directed mutagenesis kit  were used in the study.Antibodies specific for: GATA2, Control Rabbit IgG , horseradish peroxidase (HRP)-conjugated secondary antibodies , and β-actin  were used in this study. SimpleChIPRat progression-elevated gene-3 (PEG-3) was isolated from H5ts125-transformed rat embryo cancer cells by subtraction hybridization and is not expressed in normal rodent or human cells . The minpGADD34-FCGGGGTACCGAAAGAGAAAGAGAATGGGACAGCApGADD34-RCGGAAGATCT GGTCCGGTTCGGTTTGCCAAAAGCGGTCpGADD-FCGGGGTACCGAAAGAGAAAGAGAATGGGACGGCApGADD-RCGGAAGATCTGGTCCGGTTCGGTTTGCCAAAAGCGATCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACAGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGACCGAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGATCGCTTTTGGCAAACCGAACCGGACCSequences of the full-length GADD promoters with mutations are described in the The prostate cancer, pancreatic cancer, breast cancer, and neuroblastoma cell lines and primary or immortalized normal cells were plated in 24-well plates (BD Biosciences) and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The indicated cells were transfected with Luc reporter constructs pPEG-Luc, pGADD-Luc, pGAPE-Luc or a empty vector (control). Luminescence was normalized for transfection efficiency by co-transfection with a vector expressing Renilla luciferase (Luc). After 48 h of transfection, the expression level of the Luc reporter was measured by the Dual Luciferase Reporter Assay Kit (Promega) ,16.Western blotting analysis was performed as described previously ,11,64. Rwt/vol) glucose. The glucose solutions of DNA and l-PEI polymer were then mixed together to give an N:P ratio (the number of nitrogen residues of in vivo jetPEI per number of phosphate groups of DNA) of 6:1 in a total volume of 400 μL. The DNA-PEI polyplex was injected intravenously as two 200 μL injections with a 5 min interval.Low-molecular-weight l-PEI–based cationic polymer, in vivo jetPEI (Polyplus Transfection), was used for gene delivery ,27. The In vivo BLI was conducted at 24 and 48 h after the systemic delivery of reporter genes ,27. All 3 in 1% SDS. Primer pairs are listed below.ChIP assays were performed as described previously . BrieflyThe following primers were used in the ChIP assay:GADD F- GAA AGA GAA AGA GAA TGG GAC GGADD R- GTC CGG TTC GGT TTG CCA AAA GCG GGAPE F- GAA AGA GAA AGA GAA TGG GAC AGAPE R- GTC CGG TTC GGT TTG CCA AAA GCG AGAD1-1 F- GAA AGA GAA AGA GAA TGG GAC AGADD1-1 R- GTC CGG TTC GGT TTG CCA AAA GCG GGADD2-2 F- GAA AGA GAA AGA GAA TGG GAC GGADD2-2 R- GTC CGG TTC GGT TTG CCA AAA GCG AStatistical analysis was performed using SPSS 22.0 software , and statistical graphs were generated using GraphPad Prism . The generalized odds ratios (ORs) and 95% confidence interval (CI) of the promoters were calculated. The multiple comparisons were performed using false discovery rate (FDR) correction. False discovery rate (FDR) correction was used to analyze the promoter activity among the different promoters.


The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1’s molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes , and metastasis . Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration  Metastasis is a multi-step process that occurs when cells disseminate from the primary neoplasm and eventually colonize distant organs. Successful completion of this complex cascade is associated with nearly all cancer-related morbidities and mortalities. Despite its causative role in cancer-specific mortality and morbidity, a complete understanding of the process and its mechanisms remain elusive. Metastasis is regulated by three types of genes: metastasis-promoting, -suppressing, and -efficiency modifying . The proBRMS1 was discovered in the year 2000 and its re-expression in multiple cell lines significantly decreases lung metastases in syngeneic and xenograft models –8. In adBRMS1 is a known member of Sin3 histone deacetylase (HDAC) transcriptional regulatory complexes in multiple eukaryotic cell types –13. It iIn order to translate BRMS1 into clinical practice, additional structural and interactome data are necessary. This study leverages previously described features of BRMS1 to clarify its molecular functions within a cell. Previous attempts to define BRMS1 structure-function have been met with uneven success. To date no one has successfully crystallized nor determined structure for full-length, wild-type BRMS1. Computer algorithms and NMR studies have identified structural information for BRMS1 domains . BRMS1 hin vivo studies demonstrating that alteration of the domain impacts overall metastatic burden [The C-terminus of the BRMS1 protein is of particular interest as it has been shown to play a critical role in metastatic suppression, due to c burden . Of notec burden . Due to The BRMS1 primers listed in 7) were cultured into a 15 cm tissue culture plates for 24 hours then Halo-BRMS1 constructs were transfected using Lipofectamine LTX (Thermo Fisher Scientific). After 48 hours cells were harvested and washed twice with ice-cold PBS. Cells were then resuspended in mammalian cell lysis buffer (Promega) (50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% Triton® X-100, 0.1% sodium deoxycholate, 0.1 mM benzamidine HCl, 55 μM phenanthroline, 1 mM PMSF, 10 μM bestatin, 5 μM pepstatin A, and 20 μM leupeptin) followed by centrifugation at 21,000 × g for 10 min at 4°C. To remove insoluble materials, cell extracts were diluted with 700 μl of TBS  and centrifuged at 21,000 × g for 10 min at 4°C. Next, cell extracts were incubated overnight at 4°C with magnetic beads . Before elution, magnetic beads were washed four times with wash buffer . Proteins bound to magnetic beads were eluted for 2 hours at room temperature using elution buffer containing 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 0.005 mM DTT, and 2 Units AcTEV™ Protease (Thermo Fisher Scientific). The eluate was further purified by passing through a Micro Bio-Spin column  to remove residual beads prior to proteomic analyses.HEK293T cells . This application utilizes the average spectral counts of each bait across all baits to calculate the TopS score, which indicates a likelihood ratio of binding.Topological Scoring was completed for all proteins as previously described , 28. BriBRMS1 protein structure was predicted based upon its amino acid sequence retrieved from NCBI using I-TASSER , 30. BriThe primary sequence of BRMS1 was mutated at S237 to either Aspartic Acid (D) or Alanine (A). These mutants were subject to multiple predictive software applications. MuPro  utilizesThree biological replicates were performed for each bait protein . The distributed normalized spectral abundance factor (dNSAF)  was usedTo spatially map all significant 1175 proteins, we first applied a T-Distributed Stochastic Neighbor Embedding (t-SNE), a nonlinear visualization of the data followed by a k-means clustering on the two vectors generated from the t-SNE . The numTo compare the overall composition of SIN3A associated proteins between metastatic and normal breast cells, co-IP of SIN3A  from thIn order to determine the biological enrichment of differentially expressed proteins in the mutants, we subjected 1175 proteins to Reactome pathway analysis. Enrichment was completed within the R environment and several packages such as clusterProfiler, ReactomePA, DOSE and enrichplot were used.http://www.ingenuity.com, Release date: December 2016). Visualization for overlapping significant proteins was completed in Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/). Heatmaps were generated within ClustVis (https://biit.cs.ut.ee/clustvis/).Significant proteins were subjected to multiple analyses. Disease enrichment was completed within Ingenuity Pathway Analysis  cell culture medium (Thermo Fisher Scientific). MDA-MB-231 cells were cultured in 5% FBS, 0.5 mM NEAA 0 + DMEM/Sodium Bicarbonate (2.438 g/L) cell culture medium (Thermo Fisher Scientific). In both 293T and MDA-MB-231s, overexpression cells were first transduced with pENTR followed by pLENTI expression within 293FT cells according to manufacturer’s instructions (Invitrogen). Clones were selected with blasticidin. Each construct contained a single or 3x-Flag epitope tag. Expression was quantified using validated BRMS1 1a5.7 antibody (as described previously ), FLAG-M5’- GATCCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGAAGGCTAGGGCAGCTGTGTCCCCTCAGAAGAGAAAATCGGATGGACCTTGATGATGAC -3’, R: 5’ -TCGAGTCATCATCAAGGTCCATCCGATTTTCTCTTCTGAGGGGACACAGCTGCCCTAGCCTTCTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCATG -3’.F: Predicted amino acid sequences for each construct are:WTBRMS1DYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVSPQKRKSDGP1-229BRMS1DYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIS237ADYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVAPQKRKSDGPS237DDYKDDDDKMPVQPPSKDTEEMEAEGDSAAEMNGEEEESEEERSGSQTESEEESSEMDDEDYERRRSECVSEMLDLEKQFSELKEKLFRERLSQLRLRLEEVGAERAPEYTEPLGGLQRSLKIRIQVAGIYKGFCLDVIRNKYECELQGAKQHLESEKLLLYDTLQGELQERIQRLEEDRQSLDLSSEWWDDKLHARGSSRSWDSLPPSKRKKAPLVSGPYIVYMLQEIDILEDWTAIKKARAAVDPQKRKSDGP230-246BRMS1MDYKDHDGDYKDHDIDYKDDDDKKARAAVSPQKRKSDGP293T cells were grown to 90% confluency, collected and RNA extracted an RNA Isolation kit (Zymo Research). Following RNA extraction cDNA completed with the iScript cDNA synthesis kit (Bio Rad). qRT-PCR was completed with a SYBER Green assay, and cDNA loaded at a concentration of 50 ng/uL. The following primers were utilized for analysis:https://imagej.nih.gov/ij/) was utilized for analysis, followed by statistical analysis in R (‘FAS’ package), in which a Kruskal Wallis test followed by a Dunn’s adjustment was completed (https://www.R-project.org/).Cells were cultured at a seeding density of 750,000 cells/well in 5% FBS, 0.5 mM NEAA + DMEM/Sodium Bicarbonate (2.438 g/L) media in 6-well plates (Thermo Fisher). After 48 hours cells were scratched, washed, and media replaced with serum-free DMEM/Sodium Bicarbonate (2.438 g/L) (Thermo Fisher). Cells were imaged at t = 18 to 24 hrs. Image J  MDA-MB-231 cells were transduced with the construct as described above. For each construct, 3 clones were selected. 2x105 cells suspended in 100 uL of Hanks Balanced Salt Solution  were intravenously injected within the tail vein of three week-aged athymic mice (Harlan HSD athymic nude-Foxn1nu), which were restrained in a tube to facilitate tail manipulation. The lung metastases grew for 6 weeks or until the mouse was moribund and required euthanasia. Lungs were removed and fixed in a mixture of Bouin’s fixative and neutral buffered formalin (1:5 v/v), and macroscopic lung metastases were counted. This study was completed in 30 mice per construct, and 3 clones per construct. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center (#2014–2208). These experiments were performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, USDA regulations , the Federal Animal Welfare Act (7 USC 2131 et. Seq.), and the Guide for the Care and Use of Laboratory Animals, that were followed in attempts to alleviate animal suffering. A Kruskal Wallis test followed by a Dunn’s adjustment was completed as per above for statistical analysis.To measure the successful colonization of the lungs by metastases for each construct  [https://kmplot.com/analysis/) [The UALCAN web resource was utilized to assess the expression of BRMS1, BRMS1L, HDAC1, SIN3A, and SUDS3 in tumor compared to normal expression (ex.html) . All samalysis/) . PatientR packages Gplots, GGplots2, and RColor Brewer were utilized for image analysis of data.S237D ; BRMS1S237A ; BRMS11-229 ; and BRMS1230-246   . Regardl methods . These dGiven the lack of crystal structure for BRMS1 , we used the I-Tasser structure prediction method to predict the three-dimensional structure of BRMS1 using its amino acid sequence . The algin silico prediction methods, when combined with our previous findings that S237 near the C-terminus can be phosphorylated compelled us to posit that the phosphorylation site could regulate protein binding. To test this hypothesis, mass spectrometry was completed (WT), BRMS11-229, S237A, S237D, or BRMS1230-246 , applied to Z-statistics obtained from the QSPEC analysis [Mass spectrometry identified 1175 proteins whose binding was significantly (QSPEC Z-Score £-2) altered in at least one mutant compared to BRMS1analysis . This an1-229, and BRMS1230-246 cluster the most similarly. This finding may suggest that in order for BRMS1WT to interact with certain proteins it must include both phosphorylation at S237 as well as additional factors near the N-terminus that BRMS1230-246 lacks. That being said, BRMS1WT and S237D have little overlap, sharing only HDAC1, HDAC2, and SIN3A  was employed . This mend SIN3A . This coTo further examine Sin3/HDAC complex binding with BRMS1 we utilized recently published crosslinking data of complex members . BRMS1 wTo examine the interaction between SIN3A and BRMS1 further, we utilized SIN3A-endogenously expressing cells. SIN3A was co-immunoprecipitated from the nuclear lysates of immortalized, but otherwise normal, MCF-10A breast cell line, as well as within the metastatic MDA-MB-231 breast cancer cell line. These precipitates were then subjected to mass spectrometry through MALDI-TOF analysis. Interactions with SIN3A were found to be context-dependent, i.e., normal breast cell line interactions differ from metastatic cancer cell interactions, though several of these interactions have been previously published and validated in many cell lines and systems , suggest−5) with SIN3B  versus C-terminal (BRMS1230-246) binding were observed. Many of the significant pathways represented in the analysis were not represented in BRMS11-229, e.g., mRNA processing, rRNA processing, and immune responses (230-246), emphasizing the importance of the C-terminus in overall protein function and regulation of protein interactions. Importantly, many of the biological processes are tied to cancer-associated pathologies  and further study into the BRMS1 C-terminus role in these pathways could identify the molecular role of BRMS1 in metastasis. To identify binding partners distinctly affected by particular domains of BRMS1, all protein interaction changes for each mutant were considered. To ensure that putative interacting proteins were truly associated with BRMS1, an additional criterion was added, i.e., the interactor must have a high TopS value in BRMS1WT, to demonstrate an increased likelihood of binding ratio . Several of the enriched pathologies are often associated with neoplasia, including cell cycle dysregulation, cell death and survival, RNA damage repair, embryonic development, immunological disease, and infectious disease. The latter findings compliment previously published results in which re-expression of BRMS1 in metastatic MDA-MB-435 cells were highly enriched for upregulated immune response genes [To further characterize how changes in interactor binding may play a role in the development of certain pathologies, disease enrichment within Ingenuity Pathway Analysis (IPA) was completed , Src, and Fascin –71. ThesWithin these two enriched disease pathways, several proteins overlap, including BReast CAncer gene 1 (BRCA1), mechanistic target of rapamycin (mTOR), and Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) . SeveralWT and its mutants , we examined how BRMS1 interacting partners  as well Firstly, we compared the expression of the Sin3/HDAC members from normal to breast tumor samples. BRMS1, HDAC1 and SUDS3 significantly increase expression within the tumor samples, while BRMS1L has significantly decreased expression . SIN3A iTo address this speculation, we also looked for an association between one of the Sin3/HDAC members and BRMS1 expression. To do this we first separated patients by expression of each Sin3/HDAC BRMS1-interacting member by expression greater than or less than the median expression of that particular member. Within those subsets, BRMS1 expression (high vs low) was examined for patient overall survival . When loin silico prediction methods, that the C-terminus functions in protein binding, and demonstrated through mass spectrometry that the C-terminus can impact BRMS1 associations with Sin3/HDAC complexes as well as proteins outside of the complexes. We model this hypothesis in This study investigates the role of BRMS1 C-terminus in protein function, with an emphasis on the function of the S237 phosphorylation site. We hypothesized, based upon It is interesting to speculate that once expression of either BRMS1L or SIN3A is lost, it appears that BRMS1 will then function to suppress metastasis and increase survival. Our data begin to define these interactions, in particular with SIN3A, but further studies are required in order to directly define those relationships, including whether the interactions are based on phosphorylation-status to regulate metastasis. The C-terminus and phosphorylation status appear to be vital to regulating the metastasis promoting miRNA miR-10b and corresponding reductions in motility and metastasis. The data also identify specific functionalities of protein complexes previously associated with BRMS1 and metastasis.This combination of results refine the molecular impact of BRMS1 on regulating metastasis and the potential development of therapeutics based upon BRMS1.S1 FigExpression of BRMS1WT, BRMS11-229, and phospho-mutants compared to parental and vector control 293T cells. (B) qPCR quantification of BRMS1230-246 expression in 293T cells. (C) Expression of BRMS1WT and phospho-mutants compared to vector control MDA-MB-231 cells. (D). Expression of BRMS11-229 within MDA-MB-231 cells. (E) qPCR quantification of BRMS1230-246 expression in MDA-MB-231 cells.(TIF)Click here for additional data file.S2 FigBRCA data was mined in which SIN3/HDAC members were separated by median expression into those greater than the median BRMS1 expression was then examined within these patients for overall survival, with high BRMS1 (indicated by Red) and low BRMS1 (indicated by black) were accounted for. This was completed in KM Plotter.(TIF)Click here for additional data file.S3 FigBRCA data was mined in which SIN3/HDAC members were separated by median expression into those less than the median BRMS1 expression was then examined within these patients for overall survival, with high BRMS1 (indicated by Red) and low BRMS1 (indicated by black) were accounted for. This was completed in KM Plotter.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Data(DOCX)Click here for additional data file.


EPAC1) regulate obligate intracellular parasitic bacterium rickettsial adherence to and invasion into vascular endothelial cells (ECs). However, underlying precise mechanism(s) remain unclear. The aim of the study is to dissect the functional role of the EPAC1-ANXA2 signaling pathway during initial adhesion of rickettsiae to EC surfaces. Methods: In the present study, an established system that is anatomically based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae–EC adhesion. Results: The deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. Rickettsial OmpB shows a host EPAC1-dependent binding strength on the surface of a living brain microvascular EC (BMEC). Furthermore, ectopic expression of phosphodefective and phosphomimic mutants replacing tyrosine (Y) 23 of ANXA2 in ANXA2-knock out BMECs results in different binding force to reOmpB in response to the activation of EPAC1. Conclusions: EPAC1 modulates rickettsial adhesion, in association with Y23 phosphorylation of the binding receptor ANXA2. Underlying mechanism(s) should be further explored to delineate the accurate role of cAMP-EPAC system during rickettsial infection.Introduction: Intracellular cAMP receptor exchange proteins directly activated by cAMP 1 ( Rickettsia (R.), including R. rickettsii [R. parkeri [R. parkeri rickettsiosis [R. conorii, the causative agent of Mediterranean spotted fever [R. australis, which is a part of the transitional group (TRG) [R infections are frequently associated with severe morbidity and mortality [R infections, it only stops bacteria from reproducing, but does not kill the rickettsiae. A fatality rate as high as 32% has been reported in hospitalized patients with Mediterranean spotted fever [Rickettsioses are devastating human infections . These ackettsii ,3 and R. parkeri ,5,6 thatttsiosis , respected fever ; and R. up (TRG)  and causup (TRG) . A licenup (TRG) ,2,9. Typup (TRG) ,12. Untrortality ,14,15. Aed fever ,17. Comped fever ,20,21,22EPAC1, not EPAC2, is the dominant isoform [EPAC1 protected mice from fatal rickettsioses [EPAC1 suppressed rickettsial adherence to and invasion into ECs [cAMP is one of the most common and universal second messengers. cAMP regulates a myriad of important biological processes under physiological and pathological conditions. Therefore, current pharmaceutical medications target the cAMP signaling pathway more than any other . cAMP tr isoform ,39,40. Wttsioses . Our ex into ECs . HoweverANXA2), a well-characterized plasminogen and plasminogen activator receptor [Staphylococcus aureus adhesion to EC surfaces [EPAC1 modulates ANXA2 tyrosine (Y) 23 phosphorylation, and inactivation of EPAC1 suppresses ANXA2 expression on the EC luminal surface by downregulating Y23 phosphorylation [EPAC1-ANXA2 signaling pathway is involved in the regulation of rickettsial adhesion to ECs.The mechanism(s) underlying bacterial adherence to vascular endothelial cells (ECs) under shear stress from flowing blood is critical to our understanding the initial stages of the pathogenesis of endovascular bacterial infections. The constitutively expressed native proteins on and/or in the mammalian host plasma membrane play crucial functional role(s) during the initial stage of infection, just prior to EC activation. Recently we reported that endothelial surface annexin A2  provides evidence to support that EPAC1 governs rickettsial adhesion to EC surfaces in association with regulation of ANXA2 Y23 phosphorylation.In the present study, an established, anatomically based, in vivo quantitative system that measures bacterial adhesion to ECs  was combEPAC1 plays a critical regulatory role in the early stage of rickettsial invasion into nonphagocytic host cells [EPAC1-knock out (KO) mouse model [EPAC1 during rickettsial adhesion. Plaque assays reveal that the number of viable rickettsiae in circulating blood was higher in EPAC1-KO mice (n = 11) than wild-type (WT) mice (n = 10) at 30 min and 1 hr post-infection (p.i.) when the bacteria were given by the intravenous route , we found that rickettsiae mediate adhesion to the surface of ECs via host ANXA2 . CompareANXA2 .p < 0.01). To examine whether EPAC1 contributes to reOmp and BMEC binding, we compared the adhesion forces generated in WT versus EPAC1-KO BMECs; reduced adhesion forces between a reOmpB and a EPAC1-KO BMEC were recorded (p < 0.01)  microscopy [EPAC1-KO BMECs at 15 min p.i. with 10 MOI rickettsiae  . Furthercroscopy . All ECskettsiae .EPAC1 specific activator named I942 that competitively binds the cyclic nucleotide-binding domain of EPAC1 [p < 0.05) (ANXA2 in ECs diminished reOmpB binding forces to the ANXA2-KO BMEC surface (p < 0.01) , independent of its concentration in the culture medium  . Depleti < 0.01) , corroboiving EC . Interese medium .EPAC1-ANXA2 pathway is involved in the interaction of OmpB with ECs during the initial stages of bacterial binding.Collectively, these data suggest that the EPAC1 regulates endothelial luminal surface ANXA2 expression by modulating the phosphorylation of Y23 [EPAC1-targeted site in the N-terminus of ANXA2 that leads to regulation of rickettsial adhesion, we transfected ANXA2-KO BMECs with mouse WT ANXA2 construct, phosphodefective ANXA2 mutant Y23F, or phosphomimic ANXA2 mutant Y23E, respectively, which were generated by site-directed mutagenesis using their respective mutant oligonucleotides [ANXA2 in these constructs-transfected ANXA2-KO BMECs were detected, respectively (Our previous study suggested that n of Y23 . To idenleotides . Expressectively .p = 5.36 × 10−8) and phosphomimic Y23E  in ANXA2-KO BMECs rescued the diminished reOmpB and cell binding force, respectively, whereas the phosphodefective mutant Y23F (p = 0.90) failed to do so  in the plasma or other major hematological parameters [ANXA2 phosphodefective mutant Y23A, not ANXA2 phosphomimic mutant Y23E, yields a predominantly negative effect for the translocation of ANXA2 to the membrane surface. The Y23A mutant can still bind to S100A10 but only in the cytosol, not in the plasma membrane [ANXA2 functions as a binding receptor for rickettsial OmpB, in the present study we further reveal that EPAC1 regulates rickettsial adhesion to EC surfaces by targeting the ANXA2 Y23. Depletion of ANXA2 in ECs reduced the enhanced binding force between reOmpB and ECs in the presence of I942. The WT ANXA2 construct rescued EPAC1-specific activator I942-induced enhancement of reOmpB binding forces to a ANXA2-KO BMEC surface. Furthermore, Y23E mutant rescued the diminished reOmpB and cell binding force in ANXA2-KO BMEC, whereas the Y23F mutant failed. However, there was no increasing in the binding force in the phosphomimic Y23E mutant-transfected ANXA2-KO BMEC in response to I942, further suggesting that EPAC1 modulates Y23 phosphorylation of the binding receptor ANXA2. Currently the accurate pathway how EPAC1 modulates the phosphorylation of specific residues(s) in ANXA2 remains to be identified. Furthermore, the potential crosstalk between cAMP-EPAC1 system and other identified factors involved in rickettsial adhesion and/or invasion should be investigated in future studies. and S25 ,56. Phosid rafts ,57 and tartments ,58. Afteechanism . We reporameters . Specifimembrane . Given tThe colloidal probe-based AFM assay was developed to study protein–protein interactions and to quantify the interacting forces in the range from picoNewtons to nanoNewtons . During EPAC1-KO BMEC is still higher than the forces between BSA and EPAC1-KO BMEC. These data may suggest that intracellular EPAC1 is not the sole regulator on endothelial surface receptor for OmpB-mediated rickettsial binding. Such unknown host surface protein(s) remain to be identified. Furthermore, although BSA has been widely employed as a mock control using AFM or FluidFM to measure protein-protein interaction in studying bacterial adhesion [The binding forces between reOmpB and adhesion , identifEPAC1 modulates rickettsial adhesion, involving regulation of tyrosine 23 phosphorylation of the binding receptor ANXA2. This finding provides experimental and theoretical support for therapeutic strategies for rickettsiosis targeting the host cAMP-EPAC system. In conclusion, we have revealed a novel mechanism by which EPAC1-KO mice were derived as described previously [ANXA2-KO mice on the C57BL/6J background were a generous gift from Dr. Katherine Hajjar  [R. australis. Therefore, this organism was chosen as the TRG rickettsial agent of choice [All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB). Wild-type (WT) mice (C57BL/6J) were obtained from The Jackson Laboratory . C57BL/6 eviously . ANXA2-KNY, USA) . All micf choice .R. australis (strain Cutlack) was prepared as described [escribed . UninfecEPAC1-KO, and ANXA2-KO mice using established protocol [Mouse brain microvascular endothelial cells (BMECs) were isolated from wild-type, protocol ,51,62.ANXA2, ANXA2 mutant Y23E, and ANXA2 mutant Y23F were purchased from GENEWIZ .A rabbit polyclonal antibody against SFG/TRG rickettsiae was obtained from the laboratory of Dr. David Walker . AlexaFluor 594-conjugated goat anti-rabbit IgG and ProLong Gold Antifade Reagent with DAPI were purchased from Invitrogen . Recombinant rickettsial OmpB (reOmpB) (amino acids 1363–1655) was purchased from MyBioSource . Unless otherwise indicated, all reagents were purchased from Thermo Fisher Scientific. Expression constructs for GFP-tagged mouse WT ANXA2, ANXA2 Y23E, and ANXA2 Y23F constructs (1 µg/500 µL for 1 × 105 cells), respectively, using Magnetofection PolyMag and PolyMag Neo . The efficacy of transfection was evaluated with GFP fluorescent microscopy [Transfections were performed using a published protocol . Cells wcroscopy .TACGGGTCAGTCAAACCCTACACCAACTTCGATGCTGAGAGGGATGCTCTGAACATTGAGACAGCAGTCAAGACCAAAGGAGTGGATGAGGTCACCATTGTCAACATCCTGACAAACCGCAGCAATGTGCAGAGGCAGGACATTGCCTTCGCCTATCAGAGAAGGACCAAAAAGGAGCTCCCGTCAGCGCTGAAGTCAGCCTTATCTGGCCACCTGGAGACGGTGATTTTGGGCCTATTGAAGACACCTGCCCAGTATGATGCTTCGGAACTAAAAGCTTCCATGAAGGGCCTGGGGACTGACGAGGACTCCCTCATTGAGATCATCTGCTCACGAACCAACCAGGAGCTGCAAGAGATCAACAGAGTGTACAAGGAAATGTACAAGACTGATCTGGAGAAGGACATCATCTCTGACACATCTGGAGACTTCCGAAAGCTGATGGTCGCCCTTGCAAAGGGCAGACGAGCAGAGGATGGCTCAGTTATTGACTACGAGCTGATTGACCAGGATGCCCGGGAGCTCTATGATGCCGGGGTGAAGAGGAAAGGAACCGACGTCCCCAAGTGGATCAGCATCATGACTGAGCGCAGTGTGTGCCACCTCCAGAAAGTGTTCGAAAGGTACAAGAGCTACAGCCCTTATGACATGCTGGAGAGCATCAAGAAAGAGGTCAAAGGGGACCTGGAGAACGCCTTCCTGAACCTGGTCCAGTGCATCCAGAACAAGCCCCTGTACTTCGCTGACCGGCTGTACGACTCCATGAAGGGCAAGGGGACTCGAGACAAGGTCCTGATTAGAATCATGGTCTCTCGCAGTGAAGTGGACATGCTGAAAATCAGATCTGAATTCAAGAGGAAATATGGCAAGTCCCTGTACTACTACATCCAGCAAGACACCAAGGGTGACTACCAGAAGGCACTGCTGTACCTGTGTGGTGGGGATGACTGA-3’.5’-ATGTCTACTGTCCACGAAATCCTGTGCAAGCTCAGCCTGGAGGGTGATCATTCTACACCCCCAAGTGCCANXA2 mutant Y23E, the sequence of bases encoding Y23 is replaced with GAA. For ANXA2 mutant Y23F, the sequence of bases encoding Y23 is replaced with UUC.For R. australis  was injected through the tail vein, rickettsial virulence (by plaque assay) was measured in a 1 µL blood sample collected from the orbital venous sinus (OVS) at different times until 1 h post-infection (p.i.). Rickettsial antigens and mouse EPAC1 were detected with a rabbit polyclonal antibody against SFG/TRG rickettsiae (1:1000) and mouse monoclonal antibody against EPAC1 (1:500) , respectively, overnight at 4 °C, followed by incubation with AlexaFluor 594 goat anti-rabbit (1:1000) and AlexaFluor 488 goat anti-mouse (1:2000) antibodies for 15 min. Normal mouse or rabbit IgG was used as an antibody negative control during IF staining  were coated with reOmpB using published protocols ,64. Brieg-poly(ethylene glycol) (PLL-g-PEG) to block nonspecific adhesions prior to calibration using the method of Sander et al. [The FluidFM system coupling Nanosurf Core AFM  and Fluidic Pressure Controller  was used for this assay. A micropipette  (Cytosurge AG) was coated with 0.1 mg/ml poly(L-lysine)-r et al.  and pre-r et al. . The forr et al. . Ten celt-test or One-Way ANOVA . p values are as follows: ** p < 0.01 and * p < 0.05. Statistical significance is considered as p < 0.05.Values are reported as mean ± SEM. The data were analyzed using the Student’s