Prox1 represses IL-2 gene expression by interacting with NFAT2

Zhang, Shujie; Yu, Na; Wang, Linfang; Liu, Yanfeng; Kong, Yu‐Ying; Liu, Jing; Xie, Youhua

doi:10.18632/oncotarget.17278

Cited by 3 publications

(4 citation statements)

References 39 publications

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“…We also found several TFs that have not yet been associated but have been predicted to regulate differentiation in the same lineage as their target cytokines. For example, ELF3 was predicted in neutrophil differentiation based on its expression patterns during differentiation ( 84 ), and PROX1 was predicted to play a role in CD4+ T cell differentiation by suppressing key lineage cytokines ( 85 ). Overall, the lineage-associated PDI networks, generated from cytokines associated with abnormalities in lineage-specific differentiation, identified many TF interactors found by eY1H assays that have themselves been associated with differentiation of the same cell lineage.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive mapping of the human cytokine gene regulatory network

Santoso

Lal

et al. 2020

Nucleic Acids Research

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Proper cytokine gene expression is essential in development, homeostasis and immune responses. Studies on the transcriptional control of cytokine genes have mostly focused on highly researched transcription factors (TFs) and cytokines, resulting in an incomplete portrait of cytokine gene regulation. Here, we used enhanced yeast one-hybrid (eY1H) assays to derive a comprehensive network comprising 1380 interactions between 265 TFs and 108 cytokine gene promoters. Our eY1H-derived network greatly expands the known repertoire of TF–cytokine gene interactions and the set of TFs known to regulate cytokine genes. We found an enrichment of nuclear receptors and confirmed their role in cytokine regulation in primary macrophages. Additionally, we used the eY1H-derived network as a framework to identify pairs of TFs that can be targeted with commercially-available drugs to synergistically modulate cytokine production. Finally, we integrated the eY1H data with single cell RNA-seq and phenotypic datasets to identify novel TF–cytokine regulatory axes in immune diseases and immune cell lineage development. Overall, the eY1H data provides a rich resource to study cytokine regulation in a variety of physiological and disease contexts.

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Section: Resultsmentioning

confidence: 99%

Comprehensive mapping of the human cytokine gene regulatory network

Santoso

Lal

et al. 2020

Nucleic Acids Research

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“…We also found several TFs that have not yet been associated but have been predicted to regulate differentiation in the same lineage as their target cytokines. For example, ELF3 was predicted in neutrophil differentiation based on its expression patterns during differentiation (Lee et al, 2015), and PROX1 was predicted to play a role in CD4+ T cell differentiation by suppressing key lineage cytokines (Zhang et al, 2017). Overall, the lineage-associated PDI networks generated from cytokines associated with abnormalities in lineage-specific differentiation, identified many TF interactors found by eY1H assays that have themselves been associated with differentiation in the same lineage.…”

Section: Identification Of Lineage Tfs Regulating Cytokine Genesmentioning

confidence: 99%

Comprehensive mapping of the human cytokine gene regulatory network

Santoso¹,

Lal

et al. 2020

Preprint

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Proper cytokine gene expression is essential in development, homeostasis, and immune responses. Studies on the transcriptional control of cytokine genes have mostly focused on highly researched transcription factors (TFs) and cytokines, resulting in an incomplete portrait of cytokine gene regulation. Here, we use enhanced yeast one-hybrid (eY1H) assays to derive a comprehensive network comprising 1,380 interactions between 265TFs and 108 cytokine gene promoters, greatly expanding the known repertoire of TFcytokine gene interactions. We found an enrichment of nuclear receptors and confirmed their role in cytokine regulation in primary macrophages. Additionally, we used the eY1Hderived network as a framework to identify pairs of TFs that synergistically modulate cytokine gene expression, and to identify novel TF-cytokine regulatory axes in immune diseases and immune cell lineage development. Overall, the eY1H data provides a rich resource to study cytokine regulation in a variety of physiological and disease contexts. 8 High Fidelity (ThermoFisher) from a pool of human genomic DNA (Clonetech). PCR products were Gateway cloned into the pDONR P1-P4 vector and entry clones were confirmed by Sanger sequencing. Then DNA-baits were Gateway cloned upstream of two reporter genes (HIS3 and LacZ) and both reporter constructs were integrated into the yeast genome to generate chromatinized DNA-bait strains (Deplancke et al., 2006a;Deplancke et al., 2006b). Yeast DNA-bait strains were confirmed by Sanger sequencing.DNA-bait strains were mated with an array of 1,086 TF-prey yeast strains expressing human TFs fused to the yeast Gal4 activation domain (AD) (Fuxman Bass et al., 2015).Matings were performed using a Singer Rotor robotic platform that manipulates yeast strains in a 1,536-colony format. An interaction was detected when a TF-prey binds the DNA-bait and the AD moiety activates reporter gene expression, allowing the mated yeast to grow on media lacking histidine, overcome the addition of 3-amino-triazole (3AT), a competitive inhibitor of the His3p enzyme, and convert the colorless X-gal into a blue compound. Each interaction was tested in quadruplicate and interactions were considered positive if at least two of the four mated colonies tested positively (>90% of detected interactions tested positively for all four colonies).Images of mated colonies on plates lacking histidine and containing 3AT and Xgal were processed using the Mybrid web-tool to detect positive interactions (Reece-Hoyes et al., 2013). Positive interactions detected by Mybrid were then manually checked and curated. False positives detected by Mybrid, which typically occur on plates with uneven background, were removed. False negatives missed by Mybrid, which typically occur when baits exhibit high background reporter gene expression or when interactions occur next to very strong positives, were included. A total of high-quality 1,380 PDIs 9 between 265 TFs and 108 cytokine promoters were included in the final dataset (Table S3). The eY1H interactions...

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“…Prox1 regulates different target genes by forming complexes with other transcription factors, including NFATs and KLFs, in T cells and lymphatic ECs respectively. 33–35 Similarly, we identified NFAT and KLF binding sites near the V5-tagged binding sites in NFATc1 enCre Prox1 GoF VECs. Cx37, Efnb2, Flt1, and Egfl7 have known roles in either the development of the lymphatic system and cardiac valves and are highly differentially expressed in the Lymph-VEC population.…”

Section: Discussionmentioning

confidence: 71%

“…26,33 Interestingly, this revealed an enrichment of KLF and NFAT motifs, proteins previously shown to physically interact with Prox1 (Figure 4G). 34,35 Next, we sought to validate whether exemplar Prox1 bound genes were also induced by Prox1 in valve ECs. We focused on Connexin 37 (Cx37/Gja4 ), a known direct target of Prox1 in lymphatic valve ECs, which also has active enhancer signatures in wild-type VECs (Figure 4E).…”

Section: Resultsmentioning

confidence: 99%

Localized Prox1 Regulates Aortic Valve Endothelial Cell Diversity and Extracellular Matrix Stratification in Mice

O’Donnell

Gonzalez

Mukherjee

et al. 2023

ATVB

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BACKGROUND: Specialized valve endothelial cell (VEC) populations are localized oriented to blood flow in developing aortic and mitral valves, but their roles in valve development and disease are unknown. In the aortic valve (AoV), a population of VECs on the fibrosa side expresses the transcription factor Prox1 together with genes found in lymphatic ECs. In this study, we examine Prox1’s role in regulating a lymphatic-like gene network and promoting VEC diversity required for the development of the stratified trilaminar ECM (extracellular matrix) of murine AoV leaflets. METHODS: To determine whether disruption of Prox1 localization affects heart valve development, we generated mice ( NFATc1 enCre Prox1 gain-of-function) in which Prox1 is overexpressed on the ventricularis side of the AoV beginning in embryonic development. To identify potential targets of Prox1, we performed cleavage under targets and release using nuclease on wild-type and NFATc1 enCre Prox1 gain-of-function AoVs with validation by colocalization in vivo using RNA in situ hybridization in NFATc1 enCre Prox1 gain-of-function AoVs. Natural induction of Prox1 and target gene expression was evaluated in myxomatous AoVs in a mouse model of Marfan syndrome ( Fbn1 C1039G /+ ). RESULTS: The overexpression of Prox1 is sufficient to cause enlargement of AoVs by postnatal day (P)0, as well as a decrease in ventricularis-specific gene expression and disorganized interstitial ECM layers at P7. We identified potential targets of Prox1 known to play roles in lymphatic ECs including Flt1 , Efnb2 , Egfl7 , and Cx37 . Ectopic Prox1 colocalized with induced Flt1 , Efnb2 , and Cx37 expression in NFATc1 enCre Prox1 gain-of-function AoVs. Moreover, in Marfan syndrome myxomatous AoVs, endogenous Prox1, and its identified targets, were ectopically induced in ventricularis side VECs. CONCLUSIONS: Our results support a role for Prox1 in localized lymphatic-like gene expression on the fibrosa side of the AoV. Furthermore, localized VEC specialization is required for development of the stratified trilaminar ECM critical for AoV function and is dysregulated in congenitally malformed valves.

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Prox1 represses IL-2 gene expression by interacting with NFAT2

Cited by 3 publications

References 39 publications

Comprehensive mapping of the human cytokine gene regulatory network

Comprehensive mapping of the human cytokine gene regulatory network

Comprehensive mapping of the human cytokine gene regulatory network

Localized Prox1 Regulates Aortic Valve Endothelial Cell Diversity and Extracellular Matrix Stratification in Mice

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