Hdac3 regulates lymphovenous and lymphatic valve formation
Harish P. Janardhan, Zachary J. Milstone, Masahiro Shin, Nathan D. Lawson, John F. Keaney Jr., Chinmay M. Trivedi
Harish P. Janardhan, Zachary J. Milstone, Masahiro Shin, Nathan D. Lawson, John F. Keaney Jr., Chinmay M. Trivedi
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Research Article Development Vascular biology

Hdac3 regulates lymphovenous and lymphatic valve formation

Abstract

Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box–GATA–ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

Authors

Harish P. Janardhan, Zachary J. Milstone, Masahiro Shin, Nathan D. Lawson, John F. Keaney Jr., Chinmay M. Trivedi

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Figure 5

Hdac3 regulates oscillatory shear stress–mediated activation of the Gata2 intragenic enhancer.

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Hdac3 regulates oscillatory shear stress–mediated activation of the Gata...
(A) Schematic of in vitro OSS assay. (B) Transcripts for Gata2 were detected by ChIP-qPCR in scrambled shRNA– (Sc-shRNA) or Hdac3 shRNA–infected LECs subjected to static or OSS conditions and coinfected with GFP, HDAC3, HDAC3H134A,H135A, or HDAC3HEBI lentiviruses. (C) Schematics of luciferase reporter vectors composed of WT (168 bp), c.1017+512del28 (28-bp deletion, green box, 140 bp), and the c.1017+572C>T point mutation (blue box) GATA2 intragenic enhancer. (D) A dual luciferase assay was performed in LECs transfected with WT, c.1017+512del28, or the c.1017+572C>T point mutation GATA2 intragenic enhancer luciferase reporter and subjected to static or OSS conditions. Induction is represented as a ratio of firefly to Renilla luciferase activity. (E) Scramble shRNA– or Hdac3 shRNA–infected LECs, cotransfected with the WT GATA2 intragenic enhancer luciferase reporter, were subjected to static or OSS conditions. Induction is represented as a ratio of firefly to Renilla luciferase activity. Data represent the mean ± SEM and are representative of 3 independent experiments. P values were determined by Student’s t test (B and D) or by 1-way ANOVA with Sidak’s multiple comparisons test (E). See also Supplemental Figures 8 and 9.