Endothelial-specific YY1 governs sprouting angiogenesis through directly interacting with RBPJ Shuya Zhanga,b,1, Ji Young Kima,1,2, Suowen Xua, Huan Liua,b, Meimei Yina, Marina Korolevaa, Jia Guoc, Xiuying Peib, and Zheng Gen Jina,3 aAab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; bKey Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China; and cPulmonary Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 Edited by Napoleone Ferrara, University of California San Diego, La Jolla, CA, and approved January 23, 2020 (received for review September 17, 2019) Angiogenesis, the formation of new blood vessels, is tightly (11, 12). More recently, there has been an increase in the discovery regulated by gene transcriptional programs. Yin Ying 1 (YY1) is a of critical and selective transcriptional factors that mediate the ubiquitously distributed transcription factor with diverse and signaling transduction and regulate multiple aspects of EC behav- complex biological functions; however, little is known about the iors, such as sprouting (13, 14). cell-type-specific role of YY1 in vascular development and angio- Yin Yang 1 (YY1) is a ubiquitously expressed GLI-Krüppel zinc genesis. Here we report that endothelial cell (EC)-specific YY1 de- finger-containing transcription factor (15). It is highly conserved letion in mice led to embryonic lethality as a result of abnormal from Xenopus to human species and contributes to various bi- angiogenesis and vascular defects. Tamoxifen-inducible EC-specific ological processes including cell proliferation, differentiation, and YY1 YY1iΔEC knockout ( ) mice exhibited a scarcity of retinal sprout- development by functioning as a transcriptional repressor or ac- YY1iΔEC ing angiogenesis with fewer endothelial tip cells. mice also tivator in a context-dependent manner (16). Constitutive ablation displayed severe impairment of retinal vessel maturation. In an ex of YY1 in mice leads to early embryonic lethality with a defect of vivo mouse aortic ring assay and a human EC culture system, YY1 egg cylinder formation (17). A small subset of YY1 heterozygote depletion impaired endothelial sprouting and migration. Mecha- embryos is developmentally delayed and exhibits neurulation de- nistically, YY1 functions as a repressor protein of Notch signaling fects, suggesting that YY1 may have additional functions during that controls EC tip-stalk fate determination. YY1 deficiency en- late embryogenesis in mice. YY1 floxed mice showed a critical, hanced Notch-dependent gene expression and reduced tip cell for- mation. Specifically, YY1 bound to the N-terminal domain of RBPJ dose-dependent requirement for YY1 in late embryonic devel- (recombination signal binding protein for Ig Kappa J region) and opment and cell proliferation (18). It has been reported that YY1 competed with the Notch coactivator MAML1 (mastermind-like in tumor cells is implicated in tumor angiogenesis through driving – protein 1) for binding to RBPJ, thereby impairing the NICD (intra- HIF1-dependent expression of VEGF in tumor cells (19 21). cellular domain of the Notch protein)/MAML1/RBPJ complex for- However, the role of endothelial-specific YY1 in vascular devel- mation. Our study reveals an essential role of endothelial YY1 in opment and angiogenesis was unknown. To investigate the role of YY1 in the vascular system, we controlling sprouting angiogenesis through directly interacting ECKO with RBPJ and forming a YY1-RBPJ nuclear repression complex. generated EC-specific YY1-deficient (YY1 ) mice, and we YY1 | endothelial cells | sprouting angiogenesis | RBPJ | transcription factor Significance ngiogenesis represents the formation of new blood vessels Endothelial sprouting is critical for both physiological and Afrom preexisting vessels in a series of morphogenetic events pathological angiogenesis. However, the molecular mecha- that involve vascular sprouting, branching, anastomoses, lumen nisms underlying precise regulation of endothelial sprouting formation, remodeling, maturation, and final establishment of have not been fully understood. Here we show that the tran- the functionally perfused vascular network (1, 2).Vascular sprouting scription factor Yin Ying 1 (YY1) functions as a repressor of is critical for both physiological and pathological angiogenesis. Notch signaling through its inhibition of the intracellular do- Therefore, a deeper understanding of the mechanisms underlying main of the Notch protein/mastermind-like protein 1/recom- vascular sprouting is essential for developing novel therapeutic bination signal binding protein for Ig Kappa J region complex strategies to modulate angiogenesis (3, 4). Tip cells are specialized formation and modulates endothelial cell tip-stalk fate de- endothelial cells (ECs) and are required for sprouting angiogenesis. termination. Our results reveal that YY1 plays an important Tip cells directionally migrate into the avascular area with dynamic role in regulating endothelial sprouting and angiogenesis and actin-based filopodia extension (5). The filopodia are used to sense suggestYY1couldbeapotentialmoleculartargetforangiogenesis- the surroundings for guidance cues and to steer the formation of the related diseases. sprouts in a specific direction. Following tip cells sprouting, endo- Author contributions: S.Z., J.Y.K., X.P., and Z.G.J. designed research; S.Z., J.Y.K., S.X., H.L., thelial stalk cells proliferate to provide the building blocks for M.Y., and M.K. performed research; J.G. contributed new reagents/analytic tools; S.Z., growing sprouts (6, 7). J.Y.K., H.L., and Z.G.J. analyzed data; and S.Z., J.Y.K., S.X., X.P., and Z.G.J. wrote the paper. Transcription factors are essential for the proper development The authors declare no competing interest. and homeostasis of the vascular circulatory system by orchestrating This article is a PNAS Direct Submission. angiogenic gene expression (8, 9). For example, alterations in Notch Published under the PNAS license. signaling and its downstream transcription factors, such as recom- 1S.Z. and J.Y.K. contributed equally to this work. bining binding protein suppressor of hairless (RBPJ, also known as 2Present address: Division of Pharmaceutics & Comprehensive Cancer Center, College of CBF1), lead to severe impairments in vascular development and Pharmacy, The Ohio State University, Columbus, OH 43210. angiogenesis (10). Emerging evidence suggested that the interaction 3To whom correspondence may be addressed. Email: [email protected]. between transcription factors and the combinatorial regulation of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ endothelial gene transcription play a crucial role in the orientation doi:10.1073/pnas.1916198117/-/DCSupplemental. of vascular network formation and maintain endothelial integrity First published February 19, 2020. 4792–4801 | PNAS | March 3, 2020 | vol. 117 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1916198117 Downloaded by guest on September 29, 2021 ECKO flox/flox found that YY1 mice died during midgestation from vas- CreERT2; YY1 ) mice (24), a well-established model for cular defects in the yolk sac and embryo, including phenotypic inducible gene knockout in the endothelium specifically. YY1 iΔEC abnormality in sprouting angiogenesis, vascular remodeling, and was explicitly deleted in ECs of YY1 mice from postnatal day morphogenesis. To further delineate the role of endothelial YY1 2 (P2) by injecting 50 mg tamoxifen in peanut oil (Sigma) per in postnatal angiogenesis, we generated tamoxifen-inducible EC- mouse (25) (Fig. 2A), which was confirmed by isolated iΔEC iΔEC iΔEC specific YY1 knockout (YY1 ) mice. We observed that YY1 YY1 lung endothelial cells (Fig. 2B) and dual immunostain- mice exhibited abnormal retinal sprouting angiogenesis. We also ing analysis in retinal tissues (SI Appendix, Fig. S4). Then we found that YY1 depletion impaired endothelial sprouting and performed IB4 staining, which allows us to visualize the retinal iΔEC migration in intact mouse aortic ring ex vivo and in cultured vasculature. At postnatal day 5 (P5), retinas from YY1 mice human ECs in vitro. Mechanistically, we uncovered that YY1 is a showed delayed vessel growth and a hyperpruned vascular net- critical regulator of Notch signaling through directly interacting work that had fewer ECs in the retina compared with those from iΔEC with RBPJ. YY1 was found to be necessary for filopodia for- WT mice (Fig. 2C). Quantitatively, the retinas of YY1 mice mation of endothelial tip cells by regulating Notch-dependent displayed a remarkable decrease in vascular density (Fig. 2D) gene transcription. Collectively, our study reveals a role for YY1 and vessel length (Fig. 2E). Furthermore, endothelial tip cells as a crucial modulator of Notch signaling that mediates both de- were visualized by CD34 staining (26–28). Strikingly, the front iΔEC velopmental and physiological angiogenesis. vascular region of YY1 mice exhibited a blunted-end, aneurysm- like structure with fewer and malformed filopodia compared with iΔEC Results thoseinWTmice(Fig.2F). The endothelial tip cells in YY1 mice EC-Specific YY1 Deletion Leads to Embryonic Lethality Due to Vascular also had defective lumen