GATA2 is required for lymphatic vessel valve development and maintenance
Jan Kazenwadel, … , Hamish S. Scott, Natasha L. Harvey
Jan Kazenwadel, … , Hamish S. Scott, Natasha L. Harvey
Published July 27, 2015
Citation Information: J Clin Invest. 2015;125(8):2979-2994. https://doi.org/10.1172/JCI78888.
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Research Article Development Vascular biology

GATA2 is required for lymphatic vessel valve development and maintenance

Abstract

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Authors

Jan Kazenwadel, Kelly L. Betterman, Chan-Eng Chong, Philippa H. Stokes, Young K. Lee, Genevieve A. Secker, Yan Agalarov, Cansaran Saygili Demir, David M. Lawrence, Drew L. Sutton, Sebastien P. Tabruyn, Naoyuki Miura, Marjo Salminen, Tatiana V. Petrova, Jacqueline M. Matthews, Christopher N. Hahn, Hamish S. Scott, Natasha L. Harvey

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Figure 1

GATA2 Emberger mutants have reduced capacity to bind and transactivate a novel PROX1 –11 kb enhancer element.

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GATA2 Emberger mutants have reduced capacity to bind and transactivate a...
(A) Schematic demonstrating location of PROX1 –11 kb enhancer element relative to the PROX1 gene and arrangement of consensus transcription factor binding sites. (B) Treatment of hLECs with GATA2 siRNA results in substantial reduction in PROX1 levels. (C) Mutation of the GATA binding site (BS) in the PROX1 –11 kb element reduced luciferase activity mediated by WT GATA2. EV, empty vector control. Error bars correspond to ± SEM, n = 5 independent experiments. *P < 0.05, by 2-tailed Student’s t test. (D) The PROX1 –11 kb and +4.5 kb elements cooperatively enhance luciferase activity. Fold-change is shown relative to EV controls. Error bars correspond to ± SEM, n = 3 independent experiments. *P < 0.05, by 2-tailed Student’s t test. (E) HEK293 cells were cotransfected with GATA2 (WT or mutant) expression constructs together with a PROX1 –11 kb luciferase reporter construct. Luciferase activity was measured after 24 hours. Error bars correspond to ± SD (n = 6) from 2 independent experiments (1-way ANOVA, *P < 0.05 relative to WT GATA2) Emberger mutants (here and in subsequent panels) are labeled red. (F) Binding of WT and mutant GATA2 proteins to the PROX1 –11 kb element, assessed by WEMSA.