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

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.


Tamoxifen dose determination
Tamoxifen dose determinations were described previously [1]. In brief, Tamoxifen for the 100 mg/kg dose was dissolved in ethanol to yield a 100 mg/mL stock, which was then diluted with corn oil to achieve a 10 mg/ mL tamoxifen formulation (10% ethanol in final tamoxifen formulation).

Lyve1 staining
Immunohistochemical staining for Lyve1 was performed using the standard avidin-biotin-peroxidase technique. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. Endogenous peroxidase blocking was done by immersing the sections in 3% H2O2 for 15 min after which heat-induced epitope retrieval was performed using a Citrate Buffer (Biocare Medical, CA) in the Decloaker ® pressure chamber for 5 min at 120 o C. Non-specific sites were blocked by incubating slides for 20 min with 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA), endogenous biotin and avidin binding sites were blocked using Avidin-Biotin Blocking Kit (Vector Laboratories, Burlingame, CA). The sections were then incubated with goat polyclonal Lyve1 antibody (sc-19319, SCBT, Santa Cruz, CA) at a 1:400 dilution for 30 min at room temperature. For negative control tissue section, a whole molecule goat purified IgG (Chrompure, Jackson ImmunoResearch, West Grove, PA), diluted to match the protein concentration of the Lyve1 antibody was utilized. Secondary incubation was done using a biotinylated donkey anti-goat IgG antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:500 for 30 min at room temperature. Labeling incubation was done with the Vectastain RTU Kit Label (Vector Laboratories, Burlingame, CA) for 30 min at room temperature. The antigen-antibody complex was visualized using 3-diaminobenzidine (DAB) chromagen (Dako, Carpenteria, CA) for 6 min, and counterstained with modified Harris Hematoxylin. The sections were then dehydrated through graded ethanol, cleared in xylene and coverslipped.

Endomucin and Prox1 Staining
Formalin fixed, paraffin embedded mouse tissues were deparaffinized and rehydrated. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Antigen retrieval was performed with heat and pressure in a decloaking chamber, using a pH 6.0 citrate buffer retrieval solution (Biocare Medical, Concord, CA). The sections were incubated with 10% normal rabbit serum (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 20 min, followed by the avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). For Endomucin staining, sections were incubated with Endomucin (Catalog#MAB2624, Millipore, Temecula, CA) monoclonal antibody (protein G purified IgG1) and purified rat IgG1 (negative control; BD Biosciences, San Jose, CA) for 60 minutes at 1:750 dilution. Prox1 staining was performed with Prox1 (Catalog # ab174244, Abcam) antibody for 1 h at 1:50 dilution. Sections were then incubated with a biotinylated rabbit anti-rat secondary antibody (Vector Laboratories, Burlingame, CA) for 30 min at 1:500 dilution. Label incubation was performed using Vectastain Elite ABC reagent, RTU (Vector Laboratories, Burlingame, CA) for 30 min also. Antigen-antibody complex was visualized using DAB (Dako, Carpinteria, CA) for 6 min. The sections were counterstained with hematoxylin, dehydrated, cleared and cover slip applied.

Quantitative RT-PCR analysis
RNA was isolated from early embryos using the Arcturus PicoPure RNA Isolation Kit and Invitrogen RNA Isolation Kit; cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen) with Oligo dT primers. Real-Time quantitative reverse transcription PCR (qRT-PCR) measurements of individual cDNAs were performed with the SYBR Green Real-Time PCR detection system. Gene-specific primers for cell cycle regulators were designed for encoded gene transcript available at NCBI database using Primer Express. The rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer sequences are available upon request. All measurements were performed in triplicate. Values were normalized to GAPDH using the 2 -DDCt methods and expressed as ± SD. All mouse experiments were performed in accordance with NIEHS/NIH guidelines covering the humane care and use of animals in research.

Chromatin immunoprecipitation
Two litters of E8.5 mice were dissected in chilled PBS. ChIP was performed as previously described [4,5] with minor modifications. Cells were cross-linked by adding 13.5 ul of 36.6% formaldehyde per 500 ul of sample for 15 min at room temperature. Fixation was stopped, by adding 57 ul of 1.25M Glycine to the sample. Cells were lysed in cold lysis buffer 1 (50 mM HEPES-KOH, pH7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x protease inhibitors) and gently rocked at 4°C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 × g at 4°C in a tabletop centrifuge, resuspended in cold lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 × protease inhibitor), and gently rocked at 4°C for 10 min in 14ml conical tubes. Cells were pelleted at 1350 × g at 4°C in table top centrifuge and resuspended in 2 ml cold lysis buffer 3 (10 mM Tis-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-laurylsarcosine, 1 x protease inhibitors), and sonicated to 200-600 bp fragments using a Diagenode Bioruptor (3 × 10 minutes cycles, 30 s ON/ 30 s OFF at 4°C). For BRG1, and CHD4 ChIP, cells were resuspended and sonicated in sonication buffer (50mM Tris-HCl pH7.5, 140 mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.1% SDS) for 15 cycles at 30 s each on ice (18W) with 30 s off on ice between cycles. Sonicated lysate were cleared by pelleting insoluble material at 20, 000 × g at 4°C followed by incubation with antibody bound Protein G magnetic beads (2.5 ug Ab/50 ul beads / IP) in 1 ml of 0.5% BAS/PBS overnight at 4°C. Magnetic beads were washed 3 times with block buffer (0.5% BSA/PBS), incubated for approximately 4 h at 4°C with antibody in block buffer, and then washed 3 times with block buffer prior to addition of cleared cell lysates. Chromatin was immunoprecipitated using BRG1 NB100-2594; NOVUS, and IgG. Immunoprecipitated material was washed five times with cold buffer (RIPA: 50mMHEPES-KOH, pKa 7.55, 500 mM LiCl, 1mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate) and one time with TE plus NaCl 50 mM, followed by elution and reverse cross linking in 210 ul of 1% SDS in TE overnight at 65°C. 200 ul of reverse crosslinked material was treated with RNase A for 30 min to 2 h., proteinase K for 30 min to 2 h and extracted twice with phenol chloroform isoamyl alcohol, followed by ethanol precipitation with 3M sodium acetate and a glycogen coprecipitant, 80% ethanol wash and final resuspension in TE buffer or water. Nucleic acid yield was determined via Quant IT fluorescence assay (Invitrogen). Immunoprecipitated chromatin was evaluated by qPCR (Stratagene Mx300P and Brilliant SYBR Green Quantitative PCR (QPCR) master mix). Average cycle threshold amplification values and percentage of sample input were calculated. The following PCR primers are used for QPCR analysis: Lyve1 promoter sequence 1kb upstream and 1kb downstream from transcription start site (TSS) were downloaded from UCSC genome browser. Promoters of respective genes were analyzed for transcription factor binding using TRANSFAC -Gene Regulation software. Primers were designed from known transcription factor binding sites that potentially recruit BRG1.

Cell lines and cell culture
Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from the Thermal Fisher Scientific and cultured according to the instructions of the manufacturers. 2 × 10 5 HUVEC cells were seeded per well in a 6 well plate 24 hrs prior to transfection with siRNA. Next day cells were transfected with 100 pmol of either NT control or BRG1 siRNA using lipofectamine protocol according to manufacturer's instructions. Controls with lipofectamine and no lipofectamine reagent were also included. During transfection cells were maintained in complete 200PRF media supplemented with low serum growth supplement (Thermal Fisher Scientific). For endothelial cell tube formation, 24 hr post transfection cells were trypsinized and counted. 40,000 cells from each siRNA treatment were seeded on Geltrex coated well on a 24 well plate and maintained, in complete supplemented 200PRF media as described for endothelial cell tube formation assay (Thermal Fisher Scientific). Cells were incubated on the Geltrex matrix for 6 hrs and images acquired. Cells were incubated on the Geltrex matrix for 16hr and the stained with 2 ug/mL Calcein AM (Cat No C3099, Thermal Fisher) for 30 minutes followed by imaging.