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 7

Hdac3 recruits EP300 to the Gata2 intragenic enhancer in response to lymphatic shear stress.

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Hdac3 recruits EP300 to the Gata2 intragenic enhancer in response to lym...
(A and B) ChIP–qPCR analysis of H3K27 acetylation (A) and EP300 recruitment (B) to the Gata2 intragenic enhancer in scrambled shRNA– or Hdac3 shRNA–infected LECs subjected to static or OSS conditions. (C and D) Total lysates from pooled LECs subjected to static or OSS conditions were immunoprecipitated by IgG or EP300 antibody, and Western blot analysis was performed using Hdac3 antibody (C). Total Hdac3 and α-tubulin levels are shown as input controls. Hdac3 expression was quantified and normalized to total input using ImageJ software (n = 3) (D). (E) Transcripts for Gata2 were detected by ChIP-qPCR in scrambled shRNA– or EP300-shRNA–infected LECs subjected to static or OSS conditions. (F) In response to OSS, the transcription factors Tal1, Gata2, and Ets1/2 recruit Hdac3 to the Gata2 intragenic enhancer, which in turn recruits EP300 to promote Gata2 expression. In LECs lacking Hdac3, OSS failed to promote EP300 recruitment to the Gata2 intragenic enhancer, histone H3 Lys-27 acetylation, and, thereby, Gata2 expression. Data represent the mean ± SEM and are representative of 3 independent experiments. P values were determined by Student’s t test (E) or by 1-way ANOVA with Sidak’s multiple comparisons test (A, B, and D). See also Supplemental Figure 10.