Endothelial-Specific YY1 Governs Sprouting Angiogenesis Through Directly Interacting with RBPJ

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- DEVELOPMENTAL BIOLOGY late embryogenesis in mice. YY1 floxed mice showed a critical, hanced Notch-dependent gene expression and reduced tip cell formation. 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 suggest YY1 could be a potential molecular target for angiogenesis- 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

www.pnas.org/cgi/doi/10.1073/pnas.1916198117 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 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 formation, while those of WT mice had Defects. To dissect the role of YY1 in embryonic vascular devel- normal and mature actin filament bundles protruding along filopodia flox/flox opment, YY1 mice (18) were crossed with a Tie2-Cre trans- and proper lumen formation. Furthermore, we discovered that tip iΔEC genic mouse, which targets Cre recombinase in ECs as well as cells were reduced in the vascular front of YY1 mice compared iΔEC hematopoietic stem cells to generate EC-specific YY1 knockout with WT mice. The retinas of YY1 mice displayed a significant + mice (22). We observed that no viable pups expressing Tie2-Cre ; decrease in filopodia number (Fig. 2G) and filopodia extensions (Fig. flox/flox ECKO YY1 (YY1 )wereborn(SI Appendix,Table.S1). In timed 2H). Retinal angiogenesis was also moderately inhibited in endo- flox/+ mating, embryos from embryonic day 8.5 (E8.5) to E14.5 were thelial YY1 heterozygous-deficient (Cdh5-CreERT2; YY1 )mice ECKO examined. At E8.5, YY1 embryos were morphologically in- (SI Appendix,Fig.S5). These results indicate that endothelial YY1 distinguishable from wild-type (WT) embryos, but at E9.5, contributes substantially to sprouting angiogenesis and vascular net- ECKO YY1 embryos were smaller in size compared with WT em- work formation in the retina during the early postnatal period, par- ECKO bryos (Fig. 1A). At E10.5 and E11.5, the size of YY1 embryo ticularly in the formation of tip cells and endothelial sprouting. measured by crown-rump length was decreased significantly (Fig. To determine the potential role of YY1 in the maturation of 1B). Furthermore, between E12.5 and E14.5, only dead or mas- retinal vessels, YY1 was deleted in ECs from P5, and the retina sively underdeveloped embryos were observed, with most of them morphology and structure were analyzed at P12 (SI Appendix, being in the process of resorption (SI Appendix,Fig.S1). We also Fig. S6 A and B). We observed that in the retinas of ECKO iΔEC used another line of YY1 mice generated by breeding YY1 mice, there was substantially reduced vascular density, flox/flox YY1 mice with Cdh5-Cre (also known as VE-Cadherin-Cre) enlarged and tortuous vessels, a relatively sizeable avascular area transgenic mice, thereby targeting Cre recombinase in ECs (23). at the periphery, numerous microaneurysms with slight vertical + flox/flox Similarly, no viable pups expressing Cdh5-Cre ; YY1 were sprouts in the superficial vascular plexus, and little vascular born (SI Appendix,TableS2) and showed embryo development network in the deep vascular plexus (SI Appendix, Fig. S6 C–E). retarded from E9.5 to E11.5. (SI Appendix,Fig.S2). These results As a consequence, coverage of desmin (SI Appendix, Fig. S6 F indicate that the EC-specific deletion of YY1 significantly affects and G) and alpha-smooth muscle actin (α-SMA)-positive peri- early embryonic development. cytes in the retinal vessels was significantly reduced in the retinas ECKO iΔEC Detailed analysis showed that YY1 embryos exhibited of YY1 mice (SI Appendix, Fig. S6 H and I). However, the + vascular defects in the patterning of several regions of the vas- distribution pattern of collagen IV-positive (COL4 ) basement iΔEC cular tree compared with WT embryos. Whole-mount LacZ membrane along ECs in the retinal vessels of YY1 mice had + flox/flox staining of Tie2Cre ; YY1 ; ROSA26 double-transgenic no significant difference compared with that in WT mice (SI mouse embryos at E9.5 showed the absence of blood vessel Appendix, Fig. S6 J and K). Through examining mouse aortic wall iΔEC formation and disorganized and diminished vasculature com- morphology, we also observed that YY1 mice displayed much + flox/+ pared with embryos of Tie2Cre ; YY1 ; ROSA26 (Fig. 1C). thinner aortic wall with fewer ECs and fewer smooth muscle cells Whole-mount CD31 staining of embryos at E9.5 showed that in in the aortic vessel (SI Appendix, Fig. S7). Taken together, these ECKO YY1 embryos, the vascular plexus was coarse and disorga- results indicate that YY1 is critical for vessel formation and nized, and the dorsal aorta and pharyngeal arch artery were maturation during neonatal mouse development. significantly narrowed (Fig. 1D). In mutant embryos at E11.5, the vascular pattern of the head region was truncated with a Endothelial YY1 Deficiency Inhibited Sprouting Angiogenesis Ex Vivo blunted vascular front and reduced branching in mutant embryos and In Vitro. To study whether YY1 affects sprouting angiogen- A E SI Appendix A esis ex vivo, we examined the sprouting of aortic rings isolated (Fig. 1 and and , Fig. S2 ). EC markers CD31 iΔEC (Fig. 1F) and isolectin-B4 (IB4; SI Appendix, Fig. S3) immuno- from WT mice and YY1 mice. VEGF (20 ng/mL) signifi- fluorescent staining of E11.5 embryo sections showed defective cantly promoted microvessel sprouting, leading to the formation ECKO vascular network in YY1 embryos. Collectively, by using two of a vascular network in aortic rings from the WT mice. How- lines of endothelium-specific YY1 knockout mice, we observed im- ever, the sprouting capability was impaired considerably in aortic ECKO iΔEC paired vessel formation and embryonic lethality in YY1 mice, rings isolated from YY1 mice (Fig. 3 A and B) and moderately compromised in endothelial YY1 heterozygous-deficient suggesting that endothelial YY1 is essential for vascular develop- flox/+ ment during early embryonic stages. (Cdh5-CreERT2; YY1 ) mice (SI Appendix, Fig. S8 A and B). To provide further mechanistic insights into the mechanisms Endothelial YY1 Is Critical for Sprouting Angiogenesis and Vascular whereby YY1 regulates sprouting and vascular remodeling dur- Maturation. Next, we investigated the role of endothelial YY1 ing angiogenesis, we analyzed in vitro angiogenesis by primary flox/flox in postnatal angiogenesis by crossbreeding YY1 mice cultured human umbilical vein endothelial cells (HUVECs). We iΔEC with Cdh5-CreERT2 transgenic mice to generate YY1 (Cdh5- used small interference RNA (siRNA) for YY1 loss-of-function

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916198117 Zhang et al. Downloaded by guest on September 27, 2021 DEVELOPMENTAL BIOLOGY

Fig. 1. EC-specific YY1 deletion leads to embryonic lethality as a result of vascular defects. (A) Gross examination of whole embryos at the indicated stages of E9.5, E10.5, and E11.5 during the development of WT and YY1ECKO embryos. (Scale bars, 1 mm.) High-magnification images show a truncated vascular pattern of the head region at E11.5 in YY1ECKO embryos. (Scale bars, 5 mm.) (B) Quantification of embryos sizes by measuring the crown-rump length (n = 6, each group). Values represented as mean ± SEM. Paired Student’s t test was used to analyze data. *P < 0.05; **P < 0.01 vs. WT. (C) Whole-mount LacZ staining of Tie2Cre+; YY1flox/+; ROSA26 and Tie2Cre+; YY1flox/flox; ROSA26 embryos showing blue-color LacZ staining in endothelium at E9.5. (Scale bars, 1 mm; magnification panel scale bars, 5 mm.) n = 3, each group. (D) Whole-mount PECAM-1 staining analysis of WT and YY1ECKO embryos at E9.5 (Scale bars, 1 mm.) High magnification showed narrow dorsal aorta (white arrows) and pharyngeal arch artery (yellow arrowheads) in YY1ECKO embryos compared with WT. (Scale bars, 5 mm.) n = 3, each group. (E) Whole-mount PECAM-1 staining of WT and YY1ECKO embryos at E11.5. Black arrows in WT embryos showed well-organized and connected vessels in the head region, whereas black arrows in YY1ECKO embryos showed a truncated vascular pattern of the head region. (Scale bars, 1 mm; magnification panel scale bars, 5 mm.) n = 3, each group. (F) Immunofluorescent staining of the endothelial marker CD31 in WT and YY1ECKO embryos at E11.5. (Scale bars, 100 μm; 20 μm [high magnification].) (n = 3, each group.)

analysis in cultured HUVECs (Fig. 3C). YY1 depletion by the effects of YY1 knockdown in HUVECs by siRNA on the siRNA showed a remarkably reduced capacity to form spheroids expression of VEGF, VEGFR1, and VEGFR2 and phosphory- sprouting (Fig. 3 D and E) and tube formation (Fig. 3 F and G). lation of VEGF signaling downstream of ERK1/2 and AKT. We In concentration gradient experiments, through the transfection found that YY1 depletion in ECs did not affect VEGF signaling of HUVECs with different doses of YY1siRNA, we found that (SI Appendix, Fig. S9). These negative results suggest that other the degree of endothelial YY1 knockdown was linearly corre- mechanisms could be involved in endothelial YY1-mediated lated with the level of the inhibition of sprouting angiogenesis (SI angiogenesis. To understand the molecular mechanisms by Appendix, Fig. S8 C–E). Furthermore, we observed that YY1 which YY1 regulates angiogenesis, we performed an Affymetrix deletion also inhibited EC filopodia formation (Fig. 3 H–J). Microarray analysis in HUVECs treated with or without YY1 Collectively, our data indicated that YY1 positively regulates siRNA. The data showed that YY1 depletion in ECs altered sprouting angiogenesis ex vivo and in vitro. several sets of genes involved in adhesion and inflammatory re- sponses (Fig. 4A). We next used quantitative real-time PCR EC-Specific Deletion of YY1 Up-Regulates Notch-Dependent Gene (qPCR) to validate our gene array data (Fig. 4B). Interestingly, a Hey1. VEGF signaling is crucial in the regulation of endothelial category of genes related to Notch signaling was modulated after cell function and angiogenesis (29–31). To explore whether en- YY1 depletion. Specifically, hairy/enhancer-of-split related with dothelial YY1 deficiency affects VEGF signaling, we examined YRPW motif protein 1 (HEY1) was significantly increased in

Zhang et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 27, 2021 Fig. 2. Endothelial YY1 is essential for filopodia protrusion and vessel sprouting in the retina. (A) The schematic diagram for the EC-specific deletion of YY1 Δ in retinal vessels from P2 and their analyses at P5, using Cdh5-CreERT2; YY1flox/flox (YY1i EC) mice and WT mice. (B) Western blot analysis of endothelial YY1 expression in mouse lung endothelial cells isolated from WT and YY1iΔEC mice (n = 3). (C) Images of retinal whole-mount staining of the endothelial marker isolectin B4 (IB4) for WT and YY1iΔEC mice. (Scale bars, 200 μm, Left; 100 μm, Right.) n = 7, each group. Left panels were composite images from several images captured on different areas of the retina. (D and E) Quantification of retinal vascular density (D) and retinal vessel length (E)inYY1iΔEC and WT mice (n = 7, Δ each group). (F) Images of vascular sprouts at the front of the retinal vasculature in WT and YY1i EC mice. IB4 and CD34 costaining showed an aneurysm-like Δ structure with less and dysmorphic filopodia (white dotted box) in YY1i EC mice compared with WT. (Scale bars, 50 μm.) High magnification showing filopodia Δ Δ protrusions in WT and YY1i EC mice. (Scale bars, 20 μm.) (G and H) Quantification of filopodia number (G) and sprouting front length (H) in WT and YY1i EC mice. n = 7, each group. Values represent mean ± SEM. Data were analyzed using unpaired Student’s t test (D, E, G, and H). **P < 0.01 vs. WT.

YY1 siRNA-treated HUVECs compared with control siRNA- abnormal sprouting angiogenesis and vascular defects. Also, we treated HUVECs; this was further validated by western blot and observed decreased gene expression of TGF-β, PDGFD,andCCL2 immunofluorescence staining (Fig. 4 C–E). Consistent with the in YY1-depleted ECs (Fig. 4B), which could be responsible for the data in cultured ECs, Hey1 expression was also increased in reduced pericyte coverage and impaired vascular maturation in ECKO iΔEC YY1 yolk sac and embryos (SI Appendix, Fig. S10 A and B) YY1 mice. iΔEC and YY1 retina (SI Appendix, Fig. S10 C and D). Importantly, we found that siRNA depletion of HEY1 expression in HUVECs YY1 Attenuates Hey1 Expression through Directly Interacting with RBPJ rescued endothelial-YY1 knockdown-induced abnormality of and Suppressing RBPJ Activation. Notch-dependent gene transcrip- sprouting angiogenesis in vitro (Fig. 4 F–I). A pivotal role of tion is exerted through the complex ternary formation of NICD/ Notch signaling in controlling sprouting angiogenesis has been MAML1/RBPJ, in which MAML1 is a coactivator for the tran- well established (32). It has also been well found that HEY1 script factor RBPJ (34). To delineate molecular mechanisms overexpression inhibits angiogenesis (33). Thus, the alteration of whereby YY1 modulates Notch activity, we first examined the Notch target genes in YY1-deficient ECs could contribute to possible interaction between RBPJ and YY1 in HUVECs by

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916198117 Zhang et al. Downloaded by guest on September 27, 2021 coimmunoprecipitation (Co-IP). We observed that endogenous RBPJ was coimmunoprecipitated with endogenous YY1 (Fig. 5A). Reverse Co-IP experiments also consolidated the interaction between endogenous YY1 and RBPJ (Fig. 5B). Furthermore, immunofluorescent studies indicate the colocalization of YY1 with RBPJ in the nuclei of HUVECs (Fig. 5C), suggesting a bi- ologically relevant interaction between RBPJ and YY1 in ECs. To further map the interaction domain of RBPJ that associated with YY1, COS7 cells were cotransfected with expression vectors encoding full-length YY1, with either full-length RBPJ or RBPJ truncated mutants carrying a different mutation in domain structure (Fig. 5D). After the deletion of the C-terminal domain and the β-trefoil domain in RBPJ, the interaction between YY1 with RBPJ remained intact. However, this interaction was abolished after the N-terminal domain of RBPJ was deleted (Fig. 5E), suggesting that N-terminal domain (N terminus of RBPJ, amino acids 48 to 177) in RBPJ is essential for RBPJ association with YY1. It has been reported that N-terminal domain of RBPJ interacts with MAML1 (35, 36). Therefore, it is conceivable that YY1 may compete with MAML1 to bind to RBPJ, and thus affect the formation of the Notch/MAML1/RBPJ ternary complex. To test this hypothesis, we examined the effect of YY1 gain of function and loss of function on the construction of the Notch/ MAML1/RBPJ complex. We observed that overexpression of YY1 reduced the association of MAML1 and RBPJ (Fig. 5F). On the contrary, in YY1-depleted HUVECs, the interaction between MAML1 and RBPJ was significantly increased (Fig. 5G). YY1 depletion did not affect the protein expression of NICD1 and RBPJ expression in ECs (SI Appendix,Fig.S11). These results DEVELOPMENTAL BIOLOGY indicate that the YY1 suppresses the formation of the Notch/ MAML1/RBPJ complex in a competitive manner. Of note, al- though several studies have shown that the transcriptional re- pressors HDAC1/2/3 are YY1 binding proteins (37–39), we observed that overexpression of YY1 did not alter the interaction between HDAC1/2/3 with RBPJ (SI Appendix,Fig.S12). RBPJ stimulates HEY1 gene transcription by coordinating the formation of a transcriptional complex containing NICD and MAML1 (40). To provide the direct evidence showing that the interaction between YY1 and RBPJ represses NICD/MAML1/RBPJ-induced HEY1 promoter activity, we performed HEY1 luciferase activity assay. Transfection of NICD enhanced HEY1 promoter activity significantly. However, this NICD-induced elevation of HEY1 luciferase activity was suppressed by cotransfection of YY1 (Fig. 5H). Altogether, our results indicate that YY1 repressed HEY1expression via interaction with RBPJ, thus inhibiting RBPJ transcriptional activity by interfering with the formation of the NICD/MAML1/RBPJ complex.

Fig. 3. Endothelial YY1 promotes angiogenic sprouting ex vivo and in vitro. Notch Inhibition by a γ-Secretase Inhibitor Rescues Defective Sprouting iΔEC (A) Representative micrograph showed microvessel outgrowth from the Angiogenesis in YY1 Mice. To provide the direct evidence Δ aortic rings of WT and YY1i EC mice cultured in Matrigel for 7 d. (Scale bars, showing the involvement of Notch activation in defective sprout- 2 mm, Upper; 200 μm, Lower.) (B) Quantification of sprouts in aortic ring ing angiogenesis observed in YY1-deficient mice, we performed a angiogenesis assays. (n = 7, each group). (C) Western Blot analysis of the rescue experiment by treatment with DAPT, a γ-secretase in- efficiency of YY1 depletion by control siRNA, YY1siRNA after 24 h. (D and E) hibitor that indirectly inhibits Notch activation (41, 42). DAPT iΔEC Images and quantification of HUVEC spheroid sprouting after being treated (50 mg/kg body weight) was administered to YY1 mice at P3 with control siRNA or YY1 siRNA. HUVEC spheroids were embedded in col- YY1iΔEC lagen gels and stimulated with 50 ng/mL VEGF. After 48 h, the branching and P4. We observed that DAPT treatment in mice significantly rescued defective sprouting angiogenesis observed in capillary-like sprouts, which originated from the spheroid body and invaded iΔEC into the collagen matrix, were analyzed. (Scale bars, 200 μm.) n = 7, each untreated YY1 mice, which was evident by increased radial group. (F and G) Images comparing and quantifying tube formation of expansion (Fig. 6 A and B), vascular density (Fig. 6 C and D), HUVECs treated with control siRNA or YY1 siRNA. Tube formation was branch points (Fig. 6 E and F), sprout filopodia number, and assessed by labeling HUVECs with the cell-permeable green fluorescence dye filopodia length (Fig. 6 G–I). These results indicate that YY1 pro- Calcein-AM. (Scale bars, 100 μm.) n = 7, each group. (H–J) Images comparing motes sprouting angiogenesis by attenuating Notch activity in vivo. and quantifying filopodia formation when treated with control siRNA or YY1 siRNA. Filopodia formation was revealed with the actin filament marker Discussion rhodamine-phalloidin (Upper, Scale bars, 20 μm; Lower, Scale bars, 5 μm.) In this study, we revealed a crucial role of YY1 in both embry- The number of filopodia per cell was counted and expressed in a bar graph YY1 (I). The length of filopodia was quantified (J). Values represent mean ± SEM. onic and postnatal angiogenesis by using EC-specific Data were analyzed using unpaired Student’s t test (B, E, G, I, and J). n = 3, knockout mice. Mechanistically, YY1 controls sprouting angio- **P < 0.01 vs. WT or control siRNA. genesis and the formation of endothelial tip cells in the growing

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Fig. 4. YY1 regulates endothelial Hey1 expression in vitro and in vivo. (A) Transcriptional profiling analysis of YY1-dependent genes in cultured human ECs. HUVECs were treated with control siRNA andYY1 siRNA for 48 h. The gene expression profiles were analyzed by Affymetrix gene microarray. Heat map hier- archical clustering representation of microarray data highlights the expression levels of key transcription factors. (B) qPCR validated gene array data, including Notch target genes, angiogenetic genes, and vascular maturation-related genes. (C–E) Hey1 expression in HUVECs treated with control siRNA and YY1 siRNA were analyzed using qPCR (C), western blot (D), and immunofluorescent staining (E). (Scale bars, 50 μm.) n = 3. (F) Protein levels of YY1 24 h after transfection with different siRNA, analyzed via Western blot: control siRNA, YY1 siRNA, YY1 siRNA + Hey1 siRNA, Hey1 siRNA. (G) Images displaying spheroid sprouting of HUVECs treated with control siRNA, YY1 siRNA, YY siRNA+Hey1 siRNA, or Hey1 siRNA. (Scale bars, 200 μm.) (H and I) Quantification of endothelial sprout number and sprouts length from spheroid bead. Results are displayed as the mean ± SEM *P < 0.05; **P < 0.01; by one-way ANOVA. n = 7.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916198117 Zhang et al. Downloaded by guest on September 27, 2021 ABC IP TCL IP TCL YY1 RBPJ Merge IgG RBPJ IgG RBPJ IgG YY1 IgG YY1 75kd 75kd YY1 YY1 75kd 75kd RBPJ RBPJ

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Fig. 5. YY1 modulates Hey1 gene expression via direct interacting with RBPJ and suppressing RBPJ activation. (A) Co-IP with RBPJ antibody followed by western blot with the antibodies against YY1 and RBPJ in HUVECs. (B) Co-IP with YY1 antibody followed by western blot with the antibodies against YY1 and RBPJ in HUVECs. (C) Confocal microscopy shows the colocalization of endogenous YY1 with RBPJ in HUVECs. YY1 (red); RBPJ (green). (Scale bars, 20 μm.) (D) Schematic design of RBPJ deletion mutants to map YY interacting domain in RBPJ. (E) Mapping the interacting domain of RBPJ that associates with YY1 Co-IP with Flag antibody followed by western blot with the antibodies against YY1, Flag, and Tubulin in COS7 cells transfected with expression vectors encoding Flag-tagged full-length RBPJ or RBPJ deletion mutants and adenoviral YY1. (F) Co-IP with RBPJ antibody followed by western blot with the antibodies against MAML1, YY1, and RBPJ in COS7 cells transfected with NICD plasmid and infected with YY1 adenovirus. (G) Co-IP with RBPJ antibody followed by western blot with the antibodies against MAML1, YY1, and RBPJ in HUVECs treated with scrambled siRNA control and YY1 siRNA. (H) Dual-luciferase reporter assay for NICD-induced HEY1 promoter activity in COS7 cells cotransfected with a luciferase reporter plasmid encoding HEY1 gene promoter (HEY1-luciferase), in the absence or presence of NICD expressing vectors with or without adenoviral YY1 (n = 3). Values represent mean ± SEM. One-way ANOVA analyzed data. **P < 0.01; ***P < 0.001.

vascular front by regulating Notch-dependent gene expression. for regulation of angiogenesis during embryogenesis and postnatal Our study highlights the critical role of YY1 as a novel regulator development. We showed that targeted disruption of YY1 in ECs of Notch activity and angiogenesis (Fig. 6J). resulted in embryonic lethality by E12.5, with reduced vascular YY1 is a multifunctional transcription factor that can act as an network formation, abnormal blood vessels in visceral yolk sacs activator or repressor of gene expression in a context-dependent and embryos, and cardiovascular defects. The normal vasculature ECKO manner. Numerous studies have shown that tumor cell YY1 is a of YY1 embryos at E9.5 suggested that YY1 is not required crucial regulator of tumor growth and tumor angiogenesis through for de novo blood vessel formation (vasculogenesis) during early driving VEGF expression in tumor cells (43–47). For example, embryonic development. Using tamoxifen-inducible EC-specific YY1 positively regulates HIF1α-dependent VEGF expression in YY1 knockout mice, we also showed that YY1 is required for tumor cells and therefore modulates neovascularization in ma- retinal sprouting angiogenesis through controlling the formation lignant tumors (48). However, the role of endothelial YY1 in of endothelial tip cells. Importantly, we showed that the extent of vascular development and angiogenesis is unknown. In this study, YY1 knockdown correlated linearly with the severity of an- we provide the evidence showing that endothelial YY1 is critical giogenesis defects in vitro and in vivo. Collectively, our findings

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Δ Fig. 6. Inhibiting Notch activation by using DAPT rescued impaired angiogenesis in YY1i EC mice. DAPT (50 mg/kg body weight) was s.c. injected in P3 and P4 mice. At P5, the retinas were harvested and processed for isolectin B4 (IB4) whole-mount staining. The images were acquired using a confocal microscope and analyzed by Image J software for vasculature density, vessel length, filopodia number, length, and branch points. The vehicle DMSO treatment served as control. (A–D)Low magnification images and quantifications showed radial expansion (A and B), and vessel density (C and D)inYY1iΔEC mice treated by DAPT and DMSO. Left panels (A) were composite images from several images captured on different areas of the retina. (E–I) High magnification images showed the branch number in the central region of the retina (E and F) and sprouted filopodia number and filopodia length (G–I). (Scale bars, 200 μm, A;100μm; C;50μm, E.) Values represent mean ± SEM. Data were analyzed using the unpaired Student’s t test (five fields/group). **P < 0.01; ***P < 0.001. (J) Schematic diagram depicting the role of YY1 in sprouting angiogenesis. Notch target gene expression is activated by NICD binding to its transcription factor RBPJ and coactivator MAML1, which is repressed by YY1 in ECs. YY1 regulates sprouting angiogenesis by maintaining low levels of Notch-mediated gene expression in tip cells via competing for MAML1 binding to RBPJ. In the absence of endothelial YY1, Notch target genes are excessively expressed, which leads to the reduction of tip cell formation, endothelial sprouting, and angiogenesis.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1916198117 Zhang et al. Downloaded by guest on September 27, 2021 established YY1 as an essential transcription factor in the one is that tumor cell YY1 contributes to tumor angiogenesis regulation of angiogenesis. through the regulation of VEGF expression in tumor cells, the Mechanistically, we found that YY1 modulates Notch activity, other is that endothelial YY1 regulates endothelial sprouting which governs sprouting angiogenesis. The Notch signaling and tumor angiogenesis via modulating Notch signaling. Further pathway is an evolutionarily conserved pathway that plays critical studies are warranted to examine the effect of endothelial YY1 roles in controlling vascular development and EC function, such deficiency on tumor angiogenesis. as proliferation, migration, lumen formation, vessel stability, and In conclusion, our results demonstrate that YY1 is critical for cell fate determination (6, 49). A ligand/receptor interaction vascular development and angiogenesis, particularly in sprouting between neighboring cells promotes the proteolytic cleavage of angiogenesis during both embryogenesis and postnatal retinal the Notch receptor extracellular domain by the γ-secretase angiogenesis. YY1 regulates angiogenesis by maintaining low promoting the release of NICD. Subsequently, it is trans- levels of Notch-mediated HEY1 expression in tip cells via com- located to the cell nucleus, where it associates with transcrip- peting for MAML1 to bind to RBPJ. Our findings also suggest tional factor RBPJ and coactivator MAML1 (40). By doing so, that YY1 could be a potential molecular target for the treatment this triggers the transcription of downstream target genes, in- of angiogenesis-related diseases. cluding HEY/HES family members. In our study, we observed that siRNA depletion of HEY1 expression in cultured endo- Materials and Methods thelial cells rescued endothelial-YY1 knockdown-induced ab- Animals. All animal studies were reviewed and approved by the Institutional normality of sprouting angiogenesis, indicating that abnormal Animal Care and Use Committee of the University of Rochester. C57BL/6J mice (Stock No. 000664), Tie2-Cre mice (22) (Stock No. 004128), Cdh5-Cre (23) up-regulation of endothelial Notch target genes plays a crucial flox/flox role in endothelial YY1 deficiency-induced angiogenesis de- (Stock No.006137), and YY1 mice (18) (Stock No. 014649) were acquired from the Jackson Laboratory. Cdh5-CreERT2 mice (24) were obtained fects. Furthermore, we showed that YY1 directly interacted HEY1 from Ralf Adams (University of Münster), under the Material Transfer with RBPJ and YY1 represses Notch-dependent gene Agreement. Cre activity was induced in Cdh5- CreERT2 mice by administra- transcription by competing with MAML1 for binding to RBPJ. tion of tamoxifen using the following dosages and schedules: 50 μg/mouse Highlighting one contrast with our data, Yeh et al. previously tamoxifen was injected s.c. daily from postnatal day 2 (P2) to P4; 100 μg/mouse reported that YY1 inhibited Notch signaling by binding to the tamoxifen was injected daily from P5 to P7; for adult mice, 66 mg/kg tamoxifen ANK domain of Notch1 receptor (50, 51). It is possible that was intraperitoneally injected over 5 consecutive days, starting from 30 d of YY1 might modulate Notch signaling via interacting with both age (24). The mice were genotyped using DNA extracted from tail-snips (18). NICD and RBPJ. Primer sequences are listed in SI Appendix,TableS3. The formation of stable vasculature not only requires the in- DEVELOPMENTAL BIOLOGY Morphological and Histological Analysis of Embryos. Timed mating was duction of the necessary EC morphogenetic programs but also + + achieved by crossing male Cdh5-Cre; YY1flox/ (or Tie2-Cre; YY1flox/ mice) needs the recruitment of mural cell populations (vascular smooth with female YY1flox/flox mice. Embryos between E8.5 and E14.5 were dis- muscle cells or pericytes) to the abluminal wall of developing new sected in cold PBS, fixed in 4% paraformaldehyde, processed for routine vessels (52). As such, we were intrigued by the fact that endothelial paraffin embedding, sectioned at 6 μm thickness, and mounted on tissue YY1-deficient embryos and retina are unable to create a mural cell slides. Sections were stained with hematoxylin/eosin for either morphologic coat. Similar observations have been observed following the up- evaluation or immunohistochemistry. The detailed procedures are provided regulation of Notch1 activity in vivo (53). It is possible that YY1 in the SI Appendix, Materials and Methods. may induce an endothelial-mesenchymal transition program that allows ECs themselves to serve as mural cell precursors. However, Retina Whole-Mount Immunohistochemistry. Mouse pups were killed at P5 (for recent EC lineage tracing studies indicate that mural cells are sprouting angiogenesis assay) or P12 (for vascular maturation assay). Eyes recruited from the surrounding mesoderm, rather than from the were collected and fixed in 4% paraformaldehyde in PBS at room temper- ature for 2 h, and washed in PBS. Retinas were dissected and permeabilized in endothelium (54). In addition to Notch signaling, the data of our 0.3% Triton X-100 in PBS-diluted 5% goat serum at 4 °C overnight. Retinas microarray analysis showed that YY1 significantly modulated sev- β were then incubated in FITC-conjugated Isolectin B4 (1:100; L-2140; Sigma- eral genes of the paracrine signaling pathways (PDGFD, TGF- ) Aldrich) in PBS at 4 °C overnight. After washing and a brief postfixation in and inflammatory genes (CCL2, VCAM1). It is likely that YY1 4% paraformaldehyde, the retinas were either mounted flatly or processed controls mural cell recruitment in trans through paracrine signaling for multiple fluorescent labeling (25). The antibodies used in the study were pathways. Inflammatory cells such as monocytes/macrophages and listed in the SI Appendix, Materials and Methods. Flatly mounted retinas T lymphocytes also participate in the angiogenic process by se- were analyzed by fluorescent microscopy using an Olympus BX51 micro- creting inflammatory mediators and matrix metalloproteinase, scope equipped with a digital camera (Spot CCD camera) or by confocal laser which could modulate EC proliferation, migration, survival, and scanning microscopy (Olympus BX71, FluoView). Images were processed using Adobe Photoshop and analyzed by Image-J. apoptosis (55, 56). Further studies are needed to define the contributions of the alternation of paracrine signaling pathways and Statistical Analysis. All statistical analyses were calculated by GraphPad Prism inflammatory pathways to the YY1-dependent regulation of vas- 5 software (GraphPad). Mean values were compared using Student’s t test cular maturation. YY1 is ubiquitously expressed and highly con- (two groups) or one-way ANOVA (three or more groups), P value < 0.05 was served between species. Consistent with this notion, we detected the statistically significant. Numerical values were expressed as the mean ± SEM high expression of YY1 in both vascular endothelial cells and of at least three independent experiments performed in triplicate for smooth muscle cells in mouse aorta (SI Appendix,Fig.S13). Further the studies. analysis of human tissue samples also showed that YY1 is abundant A detailed description of other experimental methods and materials are in the human vascular system, especially in various endothelial cells provided in the SI Appendix, Materials and Methods. (SI Appendix,Fig.S14). Previous studies showed that YY1 in cancer cells is implicated Data Availability Statement. All data are available in the manuscript and (SI Appendix). in tumor angiogenesis through driving HIF1-dependent VEGF – expression in tumor cells (19 21). Secreted VEGF from the tumor ACKNOWLEDGMENTS. This study was supported by the National Institutes cells stimulates tumor angiogenesis. In this study, we demonstrate of Health (grants H.L.128363, H.L.130167, H.L.141171 to Z.G.J.), the Amer- a role of endothelial-specific YY1 in regulation of endothelial ican Heart Association (Grant-In-Aid 17GRNT33660671 to Z.G.J.), National sprouting and angiogenesis through YY1 directly interacting Natural Science Foundation of China (grants 81960122 and 31560290 to S.Z.), National Natural Science Foundation of Ningxia (award NZ14056 to S.Z.), RBPJ in endothelial cells. In the setting of tumor pathological and Ningxia Higher School First-class Disciplines, West China first-class angiogenesis, it is conceivable that YY1 could control tumor Disciplines Basic Medical Sciences at Ningxia Medical University (award angiogenesis and tumor growth through two distinct pathways: NXYLXK2017B07 to S.Z.).

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