Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases.

Accumulating evidence suggests that endothelial cells can be useful therapeutic targets. One of the potential targets is an endothelial cell-specific protein, Roundabout4 (ROBO4). ROBO4 has been shown to ameliorate multiple diseases in mice, including infectious diseases and sepsis. However, its mechanisms are not fully understood. In this study, using RNA-seq analysis, we found that ROBO4 downregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2. Mechanistic analysis reveals that ROBO4 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1) and TNF receptor-associated factor 7 (TRAF7), a ubiquitin E3 ligase. In this complex, ROBO4 enhances IQGAP1 ubiquitination through TRAF7, inhibits prolonged RAC1 activation, and decreases PTGS2 expression in inflammatory endothelial cells. In addition, Robo4-deficiency in mice exacerbates PTGS2-associated inflammatory diseases, including arthritis, edema, and pain. Thus, we reveal the molecular mechanism by which ROBO4 suppresses the inflammatory response and vascular hyperpermeability, highlighting its potential as a promising therapeutic target for inflammatory diseases.

Suppressive effect of black tea polyphenol theaflavins in a mouse model of ovalbumin-induced food allergy.

Food allergy is recognized as a global medical problem with increasing prevalence in recent years. Currently, the treatment of food allergy mainly involves avoidance of allergens and allergen-specific immunotherapy. Barring the spontaneous resolution of food allergy during the growth process, this disease is difficult to treat fundamentally. In recent years, the use of functional food ingredients derived from natural products has been attracting attention for their prophylactic use in food allergy. Theaflavins, i.e., black tea polyphenols, are potent antioxidants that have inhibitory effects on a variety of diseases. However, little is known about the preventive effect of theaflavins on food allergy. In this study, we designed a mouse model of food allergy and examined the effect of theaflavins using the severity of diarrhea, a symptom of food allergy, as an indicator. The administration of a black tea extract rich in theaflavins or theaflavin 1 (subgroup of theaflavins) to mice reduced the severity of diarrhea when compared with a normal diet. A reduction in malondialdehyde levels, a key marker of lipid peroxidation, was also observed. Overall, these data suggest that theaflavins may potentially inhibit food allergy by alleviating oxidative stress in the colon and can be a potential food material for prevention of food allergy.

Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Physicochemical and Biochemical Evaluation of Amorphous Solid Dispersion of Naringenin Prepared Using Hot-Melt Extrusion.

Naringenin (NRG) is a plant-derived flavonoid. Due to its antioxidant, anti-inflammatory, and analgesic activities it is beneficial to human health and is often used as a functional food ingredient; however, it has poor water solubility and low in vivo bioavailability. Therefore, the efficacy of NRG can be improved by enhancing its water solubility to increase gastrointestinal absorption. Conventional methods for the formulation of NRG are very complex and use toxic organic solvents, making them impractical for the production of functional foods. The objective of this study was to develop a safe and effective NRG-based functional food material. Previously, we established a technology to prepare amorphous solid dispersions (SDs) from functional food ingredients with poor water solubility and used hot-melt extrusion technology that is comparatively simple and does not involve the use of organic solvents. In this study, we prepared NRG SD and evaluated them both physicochemically and biochemically. NRG SD had superior water solubility and gastrointestinal absorption relative to native NRG and showed higher analgesic efficacy in rats than crystalline NRG. NRG SD was administered to mice in a mixed diet for 28 days, and organ weights and hematological/clinical biochemical parameters were assessed. NRG SD did not demonstrate severe adverse effects. The results suggest that NRG SD is a safe and highly efficacious formulation that can be used as a functional food material in the future.

Generation of KS-133 as a Novel Bicyclic Peptide with a Potent and Selective VIPR2 Antagonist Activity that Counteracts Cognitive Decline in a Mouse Model of Psychiatric Disorders.

Worldwide, more than 20 million people suffer from schizophrenia, but effective and definitive new therapeutic drugs/treatments have not been established. Vasoactive intestinal peptide receptor 2 (VIPR2) might be an attractive drug target for the treatment of schizophrenia because both preclinical and clinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Nevertheless, VIPR2-targeting drugs are not yet available. VIPR2 is a class-B G protein-coupled receptor that possesses high structural homology to its subtypes, vasoactive intestinal peptide receptor 1 (VIPR1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). These biological and structural properties have made it difficult to discover small molecule drugs against VIPR2. In 2018, cyclic peptide VIpep-3, a VIPR2-selective antagonist, was reported. The aim of this study was to generate a VIpep-3 derivative for in vivo experiments. After amino acid substitution and structure optimization, we successfully generated KS-133 with 1) a VIPR2-selective and potent antagonistic activity, 2) at least 24 h of stability in plasma, and 3) in vivo pharmacological efficacies in a mouse model of psychiatric disorders through early postnatal activation of VIPR2. To the best of our knowledge, this is the first report of a VIPR2-selective antagonistic peptide that counteracts cognitive decline, a central feature of schizophrenia. KS-133 may contribute to studies and development of novel schizophrenia therapeutic drugs that target VIPR2.

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

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.

Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells: HeyL requires Hes1 to bind diverse DNA sites.

The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-seq experiments demonstrated that, compared with HeyL alone, the HeyL-Hes1 heterodimer binds with high affinity to specific sites in the chromatin, including the binding sites of Hey1. Finally, analyses of myogenin promoter activity showed that HeyL and Hes1 act synergistically to suppress myogenic differentiation. Collectively, these results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity.

Lipin-1 is a novel substrate of protein phosphatase PGAM5.

Lipin-1 has multiple functions that regulate lipid and energy metabolism according to its subcellular localization. The subcellular localization of Lipin-1 is determined by kinase-dependent phosphorylation; however, the phosphatase that dephosphorylates and inactivates Lipin-1 has remained elusive. Using an immunoprecipitation and LC-MS/MS approach we have identified phosphoglycerate mutase family member 5 (PGAM5), a serine/threonine specific protein phosphatase, as a regulator of Lipin-1 activity. Treatment of human hepatocellular carcinoma cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which activates endogenous PGAM5, promoted dephosphorylation and nuclear accumulation of Lipin-1. Our findings further elucidate the molecular mechanisms that regulate Lipin-1.

The Robo4-TRAF7 complex suppresses endothelial hyperpermeability in inflammation.

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using Robo4-/- mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases.

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.

Endothelial Robo4 regulates IL-6 production by endothelial cells and monocytes via a crosstalk mechanism in inflammation.

Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in inflammation remain unclear. To investigate physiological Robo4 functions in inflammation, we performed a loss-of-function study in vitro and in vivo using lipopolysaccharide (LPS)-induced endotoxemia models. Subcutaneous injection of LPS into Robo4-knockout mice reduced circulating IL-6 levels. siRNA-mediated Robo4 knockdown suppressed IL-6 production induced by LPS, IL-1ß, and TNFα, in human umbilical vein endothelial cells (HUVECs). Coculture experiments with HUVECs and a monocytic cell line, U937 cells, demonstrated that Robo4 knockdown suppresses IL-6 production by both endothelial cells and U937 cells. Further coculture experiments demonstrated that Robo4 knockdown inhibited a novel IL-6 amplification mechanism mediated by crosstalk between endothelial cells and U937 cells via direct interactions and two mediators, GM-CSF and IL-1ß. Taken together, we demonstrated novel Robo4 functions in inflammation, i.e., it promotes IL-6 production by endothelial cells and immune cells via crosstalk.

Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase.

Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded through its DSGXXS motif. We show that Lipin-1 interacts with either of two E3 ubiquitin ligases, BTRC or FBXW11, and that this interaction is DSGXXS-dependent and mediates the attachment of polyubiquitin chains. Further, we demonstrate that degradation of Lipin-1 is regulated by BTRC in the cytoplasm and on membranes. These novel insights into the regulation of human Lipin-1 stability will be useful in planning further studies to elucidate its metabolic processes.

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.

Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines.

The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest.

Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity.

Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin peptidase inhibitor, clade E, member 1 (SERPINE1) by methylating H3K9. These results suggest that the ubiquitination of SETDB1 at lysine 867 controls the expression of its target gene by activating its H3K9 methyltransferase activity.

[A Method for Protein Photo-cross-linking in Living Cells Facilitating Analysis of Physiological Interactions of Proteins].

In living cells, most proteins form complexes with other proteins to exert their functions. Since protein functions are regulated in response to changes in the cellular environment, the components of the complexes can vary; therefore, proteins often interact in a weak and transient manner. To capture such labile protein interactions, we have developed a method for photo-cross-linking of proteins directly interacting in mammalian cells; this method involves expansion of the genetic code and site-specific incorporation of photoreactive amino acids into proteins. Upon cross-linking, protein complexes are stabilized by a covalent bond and can be readily isolated from cell extracts without the problems usually associated with simple affinity purification methods such as co-immunoprecipitation. Photo-cross-linkers have another benefit: they react exclusively with molecules within a range defined by the linker length. This property becomes useful for determining the binding interface of two proteins because the linkers can be introduced in a site-directed manner with our method. In this review, we first describe the expansion of the genetic code of mammalian cells for the incorporation of non-natural amino acids into proteins. Then, we introduce our recent applications and developments of the cross-linking method: identification of intracellular binding partners of the signaling protein growth factor receptor binding protein 2; analysis of the binding between membrane proteins on the cell surface; and a novel photoreactive amino acid that enables wide-ranging photo-cross-linking.

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.

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

OBJECTIVE: The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. METHODS AND RESULTS: Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. CONCLUSIONS: The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.