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. 2017 Jul 4;8(33):54925-54938.
doi: 10.18632/oncotarget.18976. eCollection 2017 Aug 15.

A role for BRG1 in the regulation of genes required for development of the lymphatic system

Ajeet Pratap Singh  1   2 , Julie Foley  3 , Arpit Tandon  4 , Dhiral Phadke  4 , H Karimi Kinyamu  1 , Trevor K Archer  1
Affiliations

A role for BRG1 in the regulation of genes required for development of the lymphatic system

Ajeet Pratap Singh et al. Oncotarget. .

Abstract

Lymphatic vasculature is an important part of the cardiovascular system with multiple functions, including regulation of the return of interstitial fluid (lymph) to the bloodstream, immune responses, and fat absorption. Consequently, lymphatic vasculature defects are involved in many pathological processes, including tumor metastasis and lymphedema. BRG1 is an important player in the developmental window when the lymphatic system is initiated. In the current study, we used tamoxifen inducible Rosa26CreERT2-BRG1floxed/floxed mice that allowed temporal analysis of the impact of BRG1 inactivation in the embryo. The BRG1floxed/floxed/Cre-TM embryos exhibited edema and hemorrhage at embryonic day-13 and began to die. BRG1 deficient embryos had abnormal lymphatic sac linings with fewer LYVE1 positive lymphatic endothelial cells. Indeed, loss of BRG1 attenuated expression of a subset of lymphatic genes in-vivo. Furthermore, BRG1 binds at the promoters of COUP-TFII and LYVE1, suggesting that BRG1 modulates expression of these genes in the developing embryos. Conversely, re-expression of BRG1 in cells lacking endogenous BRG1 resulted in induction of lymphatic gene expression in-vitro, suggesting that BRG1 was both required and sufficient for lymphatic gene expression. These studies provide important insights into intrinsic regulation of BRG1-mediated lymphatic-gene expression, and further an understanding of lymphatic gene dysregulation in lymphedema and other disease conditions.

Keywords: BRG1; LYVE1; development; lymphatic; lymphedema.

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Conflict of interest statement

CONFLICTS OF INTEREST None.

Figures

Figure 1
Figure 1. BRG1 is ubiquitously expressed in the developing embryo
Immunohistochemistry analysis demonstrates high abundance nuclear staining of BRG1 (brown stain) in brain (A), heart (B) liver (C) kidney (D) placenta (E) and in endothelial cells of the lymphatic vessel (F) of developing wild-type embryos.
Figure 2
Figure 2. BRG1 is necessary during mid-to-late stage embryogenesis
Temporal deletion of BRG1fl/fl alleles results multiple defects and led to embryonic mortality. The BRG1fl/fl pregnant female mice time mated with male BRG1fl/fl/R26CreER-TMmice were dosed with tamoxifen (100 mg/kg body weights) either at E10.5 (single dose) or at E12.5 and 13.5 (two consecutive doses). Embryo phenotype was then evaluated at E13.5 and 16.5 respectively. In E13.5 BRG1fl/fl embryos, arrows indicate normal blood vessels. In the E13.5 BRG1d/d embryos, arrows indicate defective blood vessels and hemorrhages with the asterisk indicating anemic placenta. In E16.5 BRG1d/d embryos, thin arrow indicates edema and thick arrow indicates hemorrhages.
Figure 3
Figure 3. BRG1-deficient embryos show hemorrhages
(A) Panels show gross morphology of the BRG1fl/fl and BRG1d/d embryos at the indicated developmental stages. White arrows – edema; yellow arrows – hemorrhage, lower panel of table shows number of BRG1d/d and BRG1fl/fl embryos that were analyzed for gross phenotype (B) Immunohistochemistry staining for ENDOMUCIN and LYVE1 identifies the jugular vein (jv) and the lymph sacs (ls) specifically in E13.5 BRG1fl/fl and BRG1d/d embryos. CS; carotid sinus, JLS; jugular lymphatic sac, LIJV; left internal jugular vein.
Figure 4
Figure 4. Expression level (mRNA) of a subset of lymphatic markers is attenuated in COUP-TFII (NR2F2) BRG1d/d embryos
(A) Upper panel, RNA sequencing analysis shows gene track and RNA expression signal of the LYVE1 and COUP-TFII in BRG1fl/fl vs. BRG1d/d embryos at E7.5. Lower panel, LYVE1 and COUP-TFII expression level is significantly reduced in BRG1d/d E8.5 embryos, as assessed by quantitative RT–PCR. PODOPLANIN, SOX18 and EPHRIN expression in BRG1d/d embryos is comparable to BRG1fl/fl embryos at E8.5. (B) Expression level of blood endothelial markers; TIE1, VEGFR3, CD34 and CD44 was reduced non-significantly in BRG1d/d embryos in comparison to BRG1fl/fl embryos. TIE2 expression was reduced significantly in BRG1d/d embryos. The experiment was performed with at least three biological replicates P-value < 0.05.
Figure 5
Figure 5
BRG1 is bound at the promoters of key lymphatic genes: Quantitative PCR was performed with primers designed from promoter and coding regions of the target genes (A) LYVE1 and (B) COUP-TFII after chromatin immunoprecipitation (ChIP) with BRG1 antibody and IgG using E8.5 embryos. The schematics above the QPCR data show ChIP primer locations upstream of transcription start site (TSS) and within an exon of the LYVE1 and COUP-TFII genes. The experiment was performed with at least three biological replicates P-value < 0.05.
Figure 6
Figure 6. Transient expression of BRG1 in SW13 cells affects the expression of lymphatic and blood vascular endothelial related genes
SW13 cells lacking endogenous expression of BRG1 were transiently transfected with a control empty vector or a plasmid containing full-length BRG1 cDNA. Twenty-four-hour post transfection, expression level of the lymphatic markers: LYVE1, COUP-TFII, PROX1, SOX18, EPHB2, PDPN, and VEGFR3 and blood vascular endothelial markers: TIE2, CDH5, CD44, VEGFR2 and CD34 were assessed by quantitative RT-PCR. The experiment was performed with at least three biological replicates. Data are means ± SEM of replicates. P-value < 0.05.
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