Robo4 Promoter Research Articles

A great diversity of ROBO4 expression and regulations identified by data mining and transgene mice

ROBO4 involves in the stabilization of blood vessel and mediates the migration of hematopoietic stem cell and newborn neuron. However, the patterns of expression and regulation are not quite clear. To resolve this, we analyzed the single cell sequence data, and confirmed that Robo4 mainly expresses in various endothelial cells, but also in epithelial cells, pericytes, and stem or progenitor cells of bone marrow, fibroblast cells/mesenchymal stem cell of adipose tissues, muscle cells and neuron. Robo4 expressions in endothelial cells derived from capillary vessel, tip/stalk/activated endothelial cells were higher than that in artery and large vein (matured endothelial cells). On the other hand, via mining the gene expression data deposited in the NCBI Gene Expression Omnibus database as well as National Genomics Data Center (NGDC), we uncovered that the expression of Robo4 were regulated by different stimulus and variable in diseases’ condition.Moreover, we constructed enhanced GFP (eGFP) transgene mouse controlled by Robo4 promoter using CRISPR/CAS9 system. We found GFP signals in many cell types from the embryonic section, confirming a widely expression of Robo4. Together, Robo4 widely and dynamically express in multiple cell types, and can be regulated by diverse factors.

HDAC inhibitor, MS-275, increases vascular permeability by suppressing Robo4 expression in endothelial cells

ABSTRACT Roundabout guidance receptor 4 (Robo4) is an endothelial-specific membrane protein that suppresses pathological angiogenesis and vascular hyperpermeability by stabilizing endothelial cells. Robo4 suppresses severe systemic inflammation induced by pathogens and endotoxins and inhibits tumor growth and metastasis, therefore serving as a potential therapeutic target. Although the regulation of Robo4 expression through transcription factors and epigenetic mechanisms has been studied, the role of histone deacetylases (HDACs) has not been explored. In the present study, we investigated the involvement of HDACs in the regulation of Robo4 expression. An HDAC inhibitor, MS-275, which inhibits HDAC1, HDAC2, and HDAC3, was found to suppress Robo4 expression in endothelial cells. Small interfering RNA (siRNA)-mediated knockdown of HDAC3, but not of HDAC1 and 2, also decreased its expression level. MS-275 downregulated the expression of the transcription factor complex GABP, in addition to suppressing Robo4 promoter activity. GABP expression was also downregulated by the siRNA against HDAC3. MS-275 decreased the transendothelial electrical resistance of a monolayer of mouse endothelial cells and increased the rate of leakage of Evans blue dye in the mouse lungs. In addition, MS-275 accelerated cell migration through the endothelial cell monolayer and augmented cell extravasation in the mouse lungs. Taken together, we demonstrated that MS-275 suppresses Robo4 expression by inhibiting HDAC3 in endothelial cells and enhances endothelial and vascular permeability. Thus, we demonstrated a novel mechanism regulating Robo4 expression and vascular permeability, which is anticipated to contribute to future therapies for infectious and inflammatory diseases.

PRC2 Components Maintain DNA Hypermethylation of the Upstream Promoter and Regulate Robo4 Expression in Endothelial Cells.

Roundabout4 (Robo4) is an endothelial cell-specific protein that stabilizes the vasculature in pathological angiogenesis and inflammation. We previously determined a 3-kb Robo4 promoter and demonstrated the importance of the upstream region for nuclear factor-kappaB (NF-κB)-mediated promoter activation induced by tumor necrosis factor α (TNFα). This region contains unique genomic features, including promoter region-specific DNA hypermethylation and chromatin condensation; however, the function of the region remains poorly understood. In this study, we analyzed the DNA sequences of the region and identified a motif for polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assay indicates the binding of the PRC2 component, SUZ12, to the motif. A mutation in the motif decreased DNA methylation in embryonic stem cells and increased Robo4 promoter activity in endothelial cells. An inhibitor for the PRC2 component, EZH2, induced the promoter activity and expression of Robo4 in endothelial cells treated with or without TNFα. Taken together, these results indicate that the PRC2 components maintain DNA hypermethylation and suppress Robo4 expression via the PRC2 binding motif in the upstream promoter.

ETV2-TET1/TET2 Complexes Induce Endothelial Cell-Specific Robo4 Expression via Promoter Demethylation

Although transcription factors regulating endothelial cell (EC)-specific gene expression have been identified, it is not known how those factors induce EC-specificity. We previously reported that DNA hypomethylation of the proximal promoter elicits EC-specific expression of Roundabout4 (Robo4). However, the mechanisms establishing EC-specific hypomethylation of the Robo4 promoter remain unknown. In this study, we demonstrated that the hypermethylated Robo4 proximal promoter is demethylated as human iPS cells differentiate into endothelial cells. Reporter assays demonstrated that ETV2, an ETS family transcription factor, bound to ETS motifs in the proximal promoter and activated Robo4 expression. Immunoprecipitation demonstrated direct interaction between ETV2 and methylcytosine-converting enzymes TET1 and TET2. Adenoviral expression of ETV2-TET1/TET2 complexes demethylated the Robo4 promoter and induced Robo4 expression in non-ECs. In summary, we propose a novel regulatory model of EC-specific gene expression via promoter demethylation induced by ETV2-TET1/TET2 complexes during endothelial differentiation.

Transcription factor SP1 mediates hyperglycemia-induced upregulation of roundabout4 in retinal microvascular endothelial cells

Roundabout4 (Robo4) is a gene that is expressed specifically in vasculature and is involved in the angiogenesis and integrity of blood vessels. The expression level of Robo4 increases gradually along with the development of diabetic retinopathy (DR). In this study, we explored the mechanism of transcriptional regulation of Robo4 in retinal endothelial cells, and investigated the effects of this regulation on cellular functions under hyperglycemic conditions. Human retinal endothelial cells (HREC) exposed to hyperglycemia were used to detect the expression levels of specificity protein 1 (SP1) and Robo4 by RT-qPCR and western blotting. Small interfering RNA (SiRNA) transfection technology was used to analyze the regulatory relationship between SP1 and Robo4. The effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation (ChIP) and luciferase assay. Cell migration, monolayer permeability and tube formation assays were performed to demonstrate the role of SP1/Robo4 in regulating HREC functions in hyperglycemic conditions. The results showed that hyperglycemia upregulated the mRNA and protein levels of SP1 and Robo4 in HREC. Depletion of SP1 by siRNA transfection inhibited the hyperglycemia induced overexpression of Robo4. ChIP combined with luciferase assay showed that under hyperglycemic conditions, SP1 significantly increased the transcriptional level of Robo4 via an additional SP1 binding site at −1912/−1908 in the Robo4 promoter. Repressing the SP1/Robo4 pathway effectively mitigated the abnormity in HREC migration, permeability and angiogenesis induced by hyperglycemia. All these findings indicate that hyperglycemia-induced upregulation of Robo4 is mediated by enhanced transcription of SP1. The SP1/Robo4 signaling pathway can regulate the migratory ability, monolayer permeability and angiogenesis of HREC under hyperglycemic conditions, suggesting that it may play an important role in microvascular dysfunction during DR.

A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector.

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/− (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Tumor Necrosis Factor α Induces the Expression of the Endothelial Cell-Specific Receptor Roundabout4 through the Nuclear Factor-κB Pathway.

Roundabout4 (Robo4) is an endothelial cell-specific receptor that regulates vascular stability. Recently, Robo4 has been shown to regulate vascular permeability in inflammation. However, the mechanisms regulating the Robo4 gene in the context of inflammation are poorly understood. In this study, we found that intravenous injection of tumor necrosis factor (TNF) α increased Robo4 expression in mouse organs. In vitro analyses showed that TNFα increased Robo4 expression in human primary endothelial cells, but not in cells pretreated with a nuclear factor (NF)-κB inhibitor. Reporter assays using wild-type and mutant Robo4 promoters indicated that TNFα activated the Robo4 promoter and that both the -2753 and -2220 NF-κB motifs were essential for this activation. Electrophoretic mobility shift assays demonstrated that the NF-κB p65-p50 heterodimer bound to these motifs. These findings were further supported by chromatin immunoprecipitation assays in endothelial cells. Taken together, these results indicated that TNFα induced Robo4 expression by facilitating NF-κB p65-p50 heterodimer binding to the -2753 and -2220 motifs in the Robo4 promoter in endothelial cells in the context of inflammation.

Expression of the Robo4 receptor in endothelial cells is regulated by two AP-1 protein complexes

Roundabout4 (Robo4) is an endothelial cell-specific gene that plays an important role in endothelial cell stability. We previously identified a 3-kb Robo4 promoter and demonstrated the importance of its proximal region in regulating Robo4 gene expression. To investigate the role of the upstream promoter in Robo4 gene regulation, we searched evolutionarily conserved promoter regions by phylogenetic footprinting and identified three conserved promoter regions. The most upstream region included a conserved AP-1 binding motif at position −2875. A mutation in the AP-1 motif significantly decreased Robo4 promoter activity in a transient reporter assay. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated binding of a c-Jun/c-Jun complex and a c-Jun/Fra-1 complex to the AP-1 motif. Knockdown experiments using siRNA revealed that both c-Jun/c-Jun and c-Jun/Fra-1 complexes regulate Robo4 gene expression, and that the c-Jun/c-Jun complex is essential for maximum promoter activation. Collectively, these results indicate that AP-1 complexes regulate Robo4 gene expression in endothelial cells.

Regulation of endothelial cell-specific Robo4 gene expression by DNA methylation and non-lineage specific transcription factors

Studies of tissue-specific gene expression have suggested that tissue-specific transcription factors or tissue-specific combinations of non-cell-type-specific transcription factors regulate tissue-specific gene expression. Although the studies of endothelial cell (EC)-specific gene expression has identified several transcriptional activators such as SP1, ETS family proteins, and GATA proteins, their expression and combinations are not likely to be EC-specific. To investigate the mechanism of EC-specific gene expression, we analyzed the regulation mechanism of an EC-specific gene, Roundabout4 (Robo4). We identified the 3-kb Robo4 promoter and several transcription factors including SP1 and GABP that bind to the Robo4 promoter and promote Robo4 gene expression. However, we could not explain the mechanism for EC-specific Robo4 gene expression with only those factors because their expression and combination are not EC-specific. Therefore, we hypothesized the contribution of other mechanisms, especially epigenetic control, to Robo4 gene regulation and demonstrated the importance of DNA methylation for EC-specific Robo4 expression. In this review, we summarize our recent studies and discuss the novel regulation model of Robo4 gene expression by transcription factors and DNA methylation.

Retargeting of gene expression using endothelium specific hexon modified adenoviral vector

Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.

Transcriptional Regulation of the Endothelial Cell-specific Receptor, Robo4

Roundabout4 (Robo4) is a transmembrane receptor that belongs to the Robo family of neural cell adhesion molecules. Robo4 has been shown to play a role in endothelial cell (EC) migration, proliferation, angiogenesis, and stabilizing the vasculature. Robo4 is expressed specifically in ECs in the developing embryo, placenta, tumors, and normal tissues. The goal of our study is to understand the mechanism for Robo4 gene expression. In the previous study we demonstrated that EC-specific Robo4 gene expression was regulated by the 3-kb Robo4 promoter in the 5'-flanking region of the human Robo4 gene. In vitro studies demonstrated that the Robo4 promoter is activated by the transcription factors GA-binding protein (GABP) and SP1 through the ETS binding site at -119 and the 2 SP1 binding sites at -42 and -153, respectively. The functional relevance of these sites was confirmed by in vivo reporter gene assays using Hprt locus knock in mice. In addition to the regulation mechanism by transcription factors, our recent study implicated that epigenetic modification of the promoter contributes to the Robo4 gene expression. Here I will discuss the regulation mechanism of Robo4 gene expression by transcription factors and epigenetic control.

Sox Factors Transcriptionally Regulate ROBO4 Gene Expression in Developing Vasculature in Zebrafish

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from -1170 bp to -1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.

Sox transcription factor mediated transcriptional regulation of Robo4 expression and function

Our laboratory has identified Roundabout4 (Robo4) as a bonafide angiogenic receptor. Robo4 knockdown (KD) endothelial cells show decreased migration and zebrafish (zf) embryos show intersomitic vessel (ISV) defects. We have reported that Robo4 affected cell migration results in imbalanced active Cdc42 and disrupted actin organization but precise molecules that interact with Robo4 remain unknown. To identify the proteins that bind and orchestrate Robo4 mediated angiogenesis, we performed Yeast 2 Hybrid (Y2H) screen with cytoplasmic zf Robo4 as bait and zf cDNA as prey. Y2H identified many SOX transcription factor (TF) proteins as potential binding partners. Based on their vascular specificity we focused on Sox18 and Sox7 and immune precipitation confirmed that Robo4 binds to both. In vivo, Robo4 KD in conjunction with Sox18 and Sox7 KD decreased Fli ISV expression, indicating disrupted angiogenesis. Bioinformatics on Robo4 promoter reveals Sox TF binding sites and signifies transcriptional regulation of Robo4 via Sox. Using transactivation assays we have identified that Sox18/7 temporally induce Robo4 promoter. Sox18/7 double KD decreases Robo4 transcription and expression in zf vasculature. In sum, our data indicate that Robo4 and Sox TF interact genetically and Sox TF mediated transcriptional and post‐translational Robo4 expression and function is critical in angiogenesis.Grant Funding Source: NIH

A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo

AbstractWe recently demonstrated that the 3-kb 5′-flanking region of the human ROBO4 gene directs endothelial cell–specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)–binding motif at −119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the −119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.

A Three-Kilobase Fragment of the Human Robo4 Promoter Directs Cell Type–Specific Expression in Endothelium

Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5'-flanking region directs endothelial cell-specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at -2528 and -2941, 2 SP1 consensus motifs at -42 and -153, and an ETS consensus motif at -119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA-protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (approximately 50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination. Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell-specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3-kb upstream promoter of human Robo4 contains information for cell type-specific expression in the intact endothelium.