RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1ß- and RNA-dependent co-activator of osteoclast gene expression.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

Cadherin-17 as a target for the immunoPET of adenocarcinoma.

PURPOSE: Cadherin-17 (CDH17) is a calcium-dependent cell adhesion protein that is overexpressed in several adenocarcinomas, including gastric, colorectal, and pancreatic adenocarcinoma. High levels of CDH17 have been linked to metastatic disease and poor prognoses in patients with these malignancies, fueling interest in the protein as a target for diagnostics and therapeutics. Herein, we report the synthesis, in vitro validation, and in vivo evaluation of a CDH17-targeted 89Zr-labeled immunoPET probe. METHODS: The CDH17-targeting mAb D2101 was modified with an isothiocyanate-bearing derivative of desferrioxamine (DFO) to produce a chelator-bearing immunoconjugate - DFO-D2101 - and flow cytometry and surface plasmon resonance (SPR) were used to interrogate its antigen-binding properties. The immunoconjugate was then radiolabeled with zirconium-89 (t1/2 ~ 3.3 days), and the serum stability and immunoreactive fraction of [89Zr]Zr-DFO-D2101 were determined. Finally, [89Zr]Zr-DFO-D2101's performance was evaluated in a trio of murine models of pancreatic ductal adenocarcinoma (PDAC): subcutaneous, orthotopic, and patient-derived xenografts (PDX). PET images were acquired over the course of 5 days, and terminal biodistribution data were collected after the final imaging time point. RESULTS: DFO-D2101 was produced with a degree of labeling of ~ 1.1 DFO/mAb. Flow cytometry with CDH17-expressing AsPC-1 cells demonstrated that the immunoconjugate binds to its target in a manner similar to its parent mAb, while SPR with recombinant CDH17 revealed that D2101 and DFO-D2101 exhibit nearly identical KD values: 8.2 × 10-9 and 6.7 × 10-9 M, respectively. [89Zr]Zr-DFO-D2101 was produced with a specific activity of 185 MBq/mg (5.0 mCi/mg), remained >80% stable in human serum over the course of 5 days, and boasted an immunoreactive fraction of >0.85. In all three murine models of PDAC, the radioimmunoconjugate yielded high contrast images, with high activity concentrations in tumor tissue and low uptake in non-target organs. Tumoral activity concentrations reached as high as >60 %ID/g in two of the cohorts bearing PDXs. CONCLUSION: Taken together, these data underscore that [89Zr]Zr-DFO-D2101 is a highly promising probe for the non-invasive visualization of CDH17 expression in PDAC. We contend that this radioimmunoconjugate could have a significant impact on the clinical management of patients with both PDAC and gastrointestinal adenocarcinoma, most likely as a theranostic imaging tool in support of CDH17-targeted therapies.

Wilms' tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences.

Wilms' tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3'-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.

Structure and mechanism of the mammalian fructose transporter GLUT5.

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.

Prdm16 is crucial for progression of the multipolar phase during neural differentiation of the developing neocortex.

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

Single-Dose Cisplatin Pre-Treatment Enhances Efficacy of ROBO1-Targeted Radioimmunotherapy.

We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.

Discovery of peroxisome proliferator-activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Development of a Ligand Screening Tool Using Full-Length Human Peroxisome Proliferator-Activated Receptor-Expressing Cell Lines to Ameliorate Metabolic Syndrome.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and include three subtypes (PPARα, PPARδ, and PPARγ). They regulate gene expression in a ligand-dependent manner. PPARα plays an important role in lipid metabolism. PPARγ is involved in glucose metabolism and is a potential therapeutic target in Type 2 diabetes. PPARδ ligands are candidates for the treatment of metabolic disorders. Thus, the detection of PPAR ligands may facilitate the treatment of various diseases. In this study, to identify PPAR ligands, we engineered reporter cell lines that can be used to quantify PPARγ and PPARδ activity. We evaluated several known ligands using these reporter cell lines and confirmed that they are useful for PPAR ligand detection. Furthermore, we evaluated extracts of approximately 200 natural resources and found various extracts that enhance reporter gene activity. Finally, we identified a main alkaloid of the Evodia fruit, evodiamine, as a PPARγ activator using this screening tool. These results suggest that the established reporter cell lines may serve as a useful cell-based screening tool for finding PPAR ligands to ameliorate metabolic syndromes.

Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible Peptide, Strengthening the Binding Affinity for an Antibody.

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein-protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.

Structural behavior of keratin-associated protein 8.1 in human hair as revealed by a monoclonal antibody.

Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.

Visualization of ligand-induced Gi-protein activation in chemotaxing cells.

Cell migration to chemoattractants is critically important in both normal physiology and the pathogenesis of many diseases. In GPCR-mediated chemotaxis, GPCRs transduce the gradient of an extracellular chemotactic ligand into intracellular responses via the activation of heterotrimeric G proteins. However, ligand-induced G-protein activation has not been directly imaged as yet in mammalian chemotaxing cells. We developed a Förster resonance energy transfer (FRET) probe, R10-Gi, by linking the Gi-protein α subunit to the regulator of G-protein signaling domain. The R10-Gi probe was coupled with a chemoattractant leukotriene B4 (LTB4) receptor 1 (BLT1) that induced the receptor to display a high-affinity ligand binding activity (Kd = 0.91 nM) in HEK293 cells. The R10-Gi probe exhibited an increased FRET signal in accord with the LTB4-dependent activation of Gi Furthermore, neutrophil-like differentiated human leukemia cell line 60 that expressed the intrinsic BLT1 displayed temporal Gi-protein activation in an area localized to the leading edge during chemotaxis in a shallow gradient of LTB4 These findings afford an opportunity to clarify the mechanisms underlying the subcellular regulation of Gi-protein activity, as well as GPCR-mediated ligand sensing, during chemotaxis in mammalian cells.-Masuda, K., Kitakami, J., Kozasa, T., Kodama, T., Ihara, S., Hamakubo, T. Visualization of ligand-induced Gi-protein activation in chemotaxing cells.

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active ß(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

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.

Factor H-related protein 5 interacts with pentraxin 3 and the extracellular matrix and modulates complement activation.

The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4.

Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.

Physiological Levels of Pentraxin 3 and Albumin Attenuate Vascular Endothelial Cell Damage Induced by Histone H3 In Vitro.

OBJECTIVE: Extracellular histones have strong toxicity against the vascular ECs, however, the damage is significantly attenuated in the serum. Although several plasma proteins such as albumin, APC, and PTX3 are known to inhibit the actions of histones, it is still unclear as to which plasma proteins play predominant role. The purpose of this study was to search for the major inhibitors in the serum. METHODS: ECs were cultured in serum-free medium and histone H3 was added. The effects of albumin, low-, medium-, and high-concentration of APC and PTX3 were examined by time-lapse morphological observation and by immunofluorescent staining. The treatment effects were also assessed by the cell viability assay. RESULTS: Both 5% and 2.5% albumin, medium- and high-concentration APC, and medium- and high-concentration PTX3 exerted significant protective effect. In case of damage induced by high-concentration histone H3, all of albumin, APC and PTX3 exerted effects in a concentration-dependent manner. Above results were also confirmed by the cell viability assay. CONCLUSION: Because albumin and PTX3 inhibited histone-induced damage at physiological levels found in serum, these proteins are expected to be major histone inhibitors in vivo.

G protein-dependent basal and evoked endothelial cell vWF secretion.

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.

Generation of antibodies against membrane proteins.

The monoclonal antibody has become an important therapeutic in the treatment of both hematological malignancies and solid tumors. The recent success of antibody-drug conjugates (ADCs) has broadened the extent of the potential target molecules in cancer immunotherapy. As a result, even molecules of low abundance have become targets for cytotoxic reagents. The multi-pass membrane proteins are an emerging target for the next generation antibody therapeutics. One outstanding challenge is the difficulty in preparing a sufficient amount of these membrane proteins so as to be able to generate the functional antibody. We have pursued the expression of various membrane proteins on the baculovirus particle and the utilization of displayed protein for immunization. The strong antigenicity of the virus acts either as a friend or foe in the making of an efficient antibody against an immunologically tolerant antigen. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression.

GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.