Zmiz1 is a Novel Regulator of Lymphatic Endothelial Cell Gene Expression and Function
Rajan KC, Nehal R. Patel, Anoushka Shenoy, Joshua P. Scallan, Mark Y. Chiang, Maria J. Galazo, Warrior M. Meadows
Abstract
Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.
Introduction
Lymphatic vasculature plays a crucial role in maintaining tissue fluid homeostasis, facilitating lipid absorption, and conducting immune surveillance. The proper establishment of a functional lymphatic system is vital for embryonic and postnatal development and normal function. Conversely, aberrant lymphangiogenesis is associated with various diseases, including lymphedema, diabetes, cancer, inflammation, and neurological disorders such as Alzheimer’s disease [1]. Extensive research over the years has identified several transcription factors (TFs) that regulate lymphatic development, such as PROX1, GATA2, NFATC1 and FOXC2, thereby shedding light on the role of the lymphatic vasculature in health and disease. Transcriptional co-regulators, acting as activators or repressors, play a crucial role in orchestrating selective gene transcription in a cell type-specific manner. Alterations in the interaction between TFs and co-regulators can lead to pathological conditions [2,3]. Thus, it is imperative to identify and comprehend the novel regulatory mechanisms involving transcription co-regulators during both physiological and pathological lymphangiogenesis.
Methods and materials
Mouse and treatment
All animal experiments were performed in accordance with Tulane University Institutional Animal Care and Use Committee (IACUC) policies. Zmiz1f/f [4], Prox1-CreERT2 [20] and Ai14 reporter [21] (Stock No. 007914; Jackson Laboratory) mice were crossed to generate tamoxifen inducible, lymphatic endothelial cell-specific Zmiz1 knockout mice (Zmiz1f/f;Prox1-CreERT2;Ai14). For postnatal Cre recombination, newborn offspring were administered Tamoxifen (MilliporeSigma, T5648) orally at a concentration of 100 μg on P1–P3 and mesentery lymphatic vessels were analyzed at P8. For embryonic studies, timed mating was carried out, designating E0.5 on the day a vaginal plug was observed. Mothers were administered 100 ug of tamoxifen via intraperitoneal injection at day E10.5 to induce Zmiz1 deletion in the embryonic LEC lineage.
Results
Zmiz1 is expressed in lymphatic endothelial cells
To assess whether Zmiz1 is expressed in LECs, we first performed ZMIZ1 immunofluorescent antibody staining on cultured human dermal lymphatic endothelial cells (HDLECs) and observed robust nuclear expression of ZMIZ1 (Fig 1A), similar to many other cell types (www.proteinatlas.org, www.uniprot.org, [7]). We next examined Zmiz1 expression in LECs using three publicy available single cell sequencing datasets. Firstly, data from http://betsholtzlab.org/VascularSingleCells/database.html showed robust expression of Zmiz1 in LECs associated with murine lung ECs [26,27] (Fig 1B). Secondly, utilizing a single cell atlas of adult mouse mesenteric LECs [28], we observed Zmiz1 expression in collecting, pre-collecting, capillary, and valve LECs (Fig 1C). Finally, we found varying levels of Zmiz1 expression in different LEC subtypes surveyed via a single-cell transcriptional roadmap of the mouse and human lymph nodes [29] (S1 Fig). Overall, these expression datasets show that Zmiz1 is expressed in several subtypes of LECs, thereby establishing its presence in the lymphatic endothelium and indicating a potential role for Zmiz1 in lymphatic vessel development.
Discussion
The role of Zmiz1 in lymphatic endothelial cells and lymphatic development is unknown [4–6,8,18]. In this study, we utilized an inducible Prox1-CreERT2 mouse model to investigate the effects of Zmiz1 loss of function in vivo, as well as using siRNA knockdown of ZMIZ1 in vitro in HDLECs. Our transcriptional profiling of HDLECs with Zmiz1 knockdown revealed impaired expression of migration, proliferation, and lymphatic vessel development genes, with a significant reduction in the expression of PROX1—a master regulator of lymphatic vasculature development (Figs 1 and 2). We also found that Zmiz1 facilitates chromatin accessibility in PROX1 genomic regulatory regions to regulate its expression. Furthermore, our mouse model showed a significant reduction in mesenteric lymphatic vessel valves upon Zmiz1 ablation (Fig 4). These findings suggest a potential role for Zmiz1 in lymphatic development, warranting further investigation.
Acknowledgments
We thank Dr. Jovanny Zabaleta and Dr. Jone Garai at The Louisiana Cancer Research Consortium (LCRC) Translational Genomics Core Center for their continued support with our RNA-Seq and ATAC-Seq experiments.
Citation: K. C. R, Patel NR, Shenoy A, Scallan JP, Chiang MY, Galazo MJ, et al. (2024) Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function. PLoS ONE 19(5): e0302926. https://doi.org/10.1371/journal.pone.0302926
Editor: Kenji Tanigaki, Shiga Medical Center, JAPAN
Received: August 25, 2023; Accepted: April 15, 2024; Published: May 8, 2024
Copyright: © 2024 K. C. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data and Supporting Information are within the manuscript and are available in Gene Expression Omnibus (GEO) database under the following accession numbers: GSE225057 and GSE225130.
Funding: This work was supported by Tulane University start-up funds (SMM), NIH-R01 HL139713 (SMM), NIH-R01 HL163196 (SMM), NIH-R01 NS128106 (MPG), NIH-R01 HL142905 (JPS), NIH-R01 AI136941(MYC) and the Priddy Spark Fund -Tulane University (SMM and MJG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0302926#abstract0
