Integrative Networks by BRWD3 Regulates Cytoskeleton Organization and Cell Motility

Background Eukaryotic cytoskeleton forms and keeps cell shape, transports intracellular particles and organelles, determines cell motility and other important cellular events. A large number of regulators of cytoskeleton organization have been identied, but the detailed regulatory mechanism still remains obscure. Previous reports suggest that BRWD3 may be a regulator of cytoskeleton organization in Drosophila melanogaster, and inuences cell shape. Therefore, we investigated the molecular network of BRWD3 regulating cytoskeleton organization. this study, we observed the alteration of cell cell motility, and proliferation when BRWD3 was knocked down in MCF-7 and MDA-MB-231 cell lines. The cells were rounded, cell decreased when BRWD3 was Using chromatin immunoprecipitation combining with we found that BRWD3 inuenced the cytoskeleton organization, cell shape, and cell motility through regulating expression of the cytoskeleton associative genes including ARF1, ABI2, ARPC3, ARPC1A, RHOC, MEF2C, and VIM.


Background
The dynamic organization of the eukaryotic cytoskeleton determines cell shape, transportation of intracellular particles and organelles, cell motility, and plays critical roles in embryonic development, homeostasis, and disorders [1]. Regulating cytoskeleton organizations have been focused on in the models including C. elegans [2,3] and Drosophila [4,5]. A large number of regulators have been identi ed, such as FAM40A, FAM40B, Rac2, and BRWD3 [5]. Both FAM40A and FAM40B regulate stress bers and angiogenic loop formation [6]. Rac2 encoding small GTPase regulates the actin cytoskeleton and controls salivary gland migration [7]. We still don't know about the role of BRWD3 in the cytoskeleton organization.
BRWD3 (Bromodomain and WD Repeat Domain Containing 3), also is known as FLJ38658 or BRODL.

Results
Previously, the alterations of cell morphology and cytoskeleton organization are observed in PC3, S2R+, and HeLa cell lines when BRWD3 was depleted [4,5]. With the method of cell immuno uorescence, we observed that number of protrusion decreased, cellular shape rounded and smaller, and spread area After the cytoskeleton alteration was observed in MDA-MB-231 cell lines when BRWD3 was knocked down, we resolved the regulation mechanism of BRWD3 on cytoskeleton organization and cell motility. ChIP-Seq was used to explore an integrative molecular network of BRWD3. The quality of the raw data was checked, and the clean data was matched to the Ensemble GRCh38.81. We identi ed 5491 ChIP-seq peaks (-10*log 10 (p-value)>50). The 2939 unique genes were annotated and enriched with the GENEONTOLOGY (http://geneontology.org/). With the Fisher's exact test and the Bonferroni correction for multiple testing, the uniquely mapped genes were annotated with the biological process (GO: 0008150) and enriched. 264 genes were enriched with the cytoskeleton organization (GO: 0007010, p= 3.4E-07), and 131 genes were enriched with the regulation of cytoskeleton organization (GO: 0051493, p= 0.00505). 194 genes were enriched with the regulation of cell motility (GO: 2000145, p= 0.0207), and 156 genes were enriched with cell morphogenesis (GO: 0000902, p= 0.0386) ( Figure 6). Six genes, including NRP1, PTK2, ABL1, CDK5, EVL, and ARHGEF2, were identi ed among the four groups, including regulation of cell morphogenesis, cytoskeleton organization, cell motility, and regulation of cytoskeleton organization.
From a ChIP-seq experiment, the identi cation of sites was enriched, and not be puri ed. To measure effective genes whose functions are enriched with the regulation of cytoskeleton organization, cell morphogenesis, and cell motility, and whose promoters are a nity with BRWD3, we analyzed the regulation relationship between BRWD3 and its binding genes using real-time quantities PCR. We excluded ABL1 and EVL because they were bound to BRWD3, not through promoters. From the results of gene annotation, several critical genes related to cytoskeleton organization were also selected, including ARF1, ABI2, ARPC3, ARPC1A, RHOC, MEF2C, and VIM. When BRWD3 was knocked down in MCF-7 and MDA-MB-231 cell lines (Figure 7 A), the transcription level of most of the selected genes, including PTK2, ARF1, ABI2, ARPC3, ARPC1A, RHOC, VIM, CDK5, MEF2C, and ARHGEF2, were in uenced signi cantly (p<0.05) (Figure 7 B, C). These 10 genes might be important nodes with a close relationship between BRWD3 and cell shape, cytoskeleton organization, and cell motility.

Discussion
Previous reports suggest that BRWD3 be a regulator of actin lament and microtubules, based upon cell shape and actin phenotype alteration when dBRWD3 was knocked down [4,5]. In this study, we have provided a novel insight into the molecular network of BRWD3, which in uences actin laments-and microtubule-based organization, cell shape and motility, and may lay the foundation for understanding the pathogenesis of complex disorders including mental retardation X-linked 93 and relative carcinoma phenotype.
Previously, dBRWD3 encoding protein is identi ed as a dominant suppressor of hedgehog loss-offunction in the developing eye. Partial loss-of-function for the dBRWD3 leads to small rhabdomeres in photoreceptor cells. It is the rst article to report that dBRWD3 function is involved in cell shape regulation in Drosophila [8]. The dBRWD3, clustered together with ZMYM6, Cdc42, WTIP, BRWD1, HYPC, and EPB41l4A, in uences peripheral actin, microtubule process, and cell shape in Drosophila cell lines [4,5]. However, how to regulate cytoskeleton organization remains obscure. In this study, we observed that alteration of protrusion structures, cell shape, and cell movements in either MCF-7 or MDA-MB-231 cell lines, when BRWD3 was knocked down. Subsequently, the BRWD3 network regulating the cytoskeleton organization was screened with ChIP-seq. The genes selected in this study were enriched with the regulation of cytoskeleton organization and bound to BRWD3 through their promoters, including PTK2, ARF1, ABI2, ARPC3, ARPC1A, RHOC, VIM, CDK5, MEF2C, and ARHGEF2. The transcription level of these 10 genes was changed signi cantly when BRWD3 was knocked down. During the cytoskeleton organization process, the proteins of ARPC3 and ARPC1A are involved in the formation of the actin-related protein 2/3 (Arp2/3) protein complex, and the proteins of VIM, ARHGEF2, CDK5, ARPC3 are involved in the process of polymeric cytoskeletal ber. All of these molecules are involved in the regulation of actin polymerization and mediates the formation of branched actin networks. The collective results provided a potential possibility that BRWD3 was a modulator of cytoskeleton organization through regulating on actin laments, microtubule, and intermediate laments, and engaged extensive cross-talk among their organization.

Conclusions
In summary, the molecular network by BRWD3 may underlay the molecular mechanism on alteration of cytoskeleton reorganization, cell shape and motility. The cell shape changes due to microtubules contact the grid and tensile actomyosin cortex rounded-up underneath the plasm membrane [26]. Our observations support the view that the cytoskeleton should be considered as a uni ed system to exert its functions in the development and pathogenesis of complex disorders [27]. Although the view is supported by microscopic observation and cell motility assay in cultured living cells, the underlying precise mechanism still needs to study furthermore in cytoskeleton biology. referring to protocol. Opti-MEM (Invitrogen) was used as the transfection medium. RNA interference was carried out by transfecting 50 nM control and si-BRWD3 respectively, from GenePharma Company (China). Silencer si-BRWD3 sequence was followed as: GCAAACGUCAGCAUACUUATT. Scrambled siRNA (UUCUCCGAACGUGUCACGUTT) was used as control. All these assays were carried out at least in triplicate.

Cell immuno uorescence
The cells were plated on slides. The next day, cells were infected to silence BRWD3. After 48 h of transfection, cells were xed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with 0.05% Triton-X for 3 min, and then blocked with 2% bovine serum albumin for 1 h. The antibodies including FITC-labeled Phalloidin (#40735ES75, YIASEN Biotech Co.Ltd, China), β-tublin (ab6046, USA) were used to show micro lament and microtubules in the cells. After sections were washed with phosphate-buffered saline (PBS), 3×5 min, the cells were then counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei labeling. Images were collected by an Olympus uorescence microscope at ×40 magni cation.

Detection of protein expression with western blot
The cells were washed with cold PBS and then were harvested with RIPA buffer containing proteinase inhibitors. The protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene uoride After incubation with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature, the membrane was washed with PBST again, 3×10 min. Then, the proteins were detected and exhibited with ECL reagents (Pierece, USA).

Cell motility
To measure the motility ability of cells, scratch wound assay and transwell assays were carried out. For scratch wound assay, breast cancer cells were cultured into a 6-well plate. After 24 hours of RNAi, the wound was scraped with a sterile pipette tip. The 6-well plate was washed twice to clean the cells fallen off. The cells were cultured with media as above-mentioned, except with 5% FBS. The images were obtained at 0 h, 24 h, and 48 h, and analyzed with Image J. The cell motility ability was measured and evaluated with the scratch wound healing area.
To do transwell assays, according to a previous report with modi cation [28], the mixture of serum-free media and matrigel (Corning, NY, USA) was put into the insert chamber (BD Falcon, Franklin Lakes, NJ, USA) overnight. The 5×10 4 of control cells (siRNA-Control) and treated (si-BRWD3) of MCF-7 or MDA-MB-231 were digested and re-suspended in serum-free DMEM and seeded into the insert. The bottom chamber was lled with 600 µl of DMEM plus 10% FBS. After migration for 48 h, non-migration cells on the upper side of the lter were scraped off with a cotton swab, and cells on the bottom were xed with 4% PFA for 10 min, and then stained with 0.2% crystal violet in 20% methanol for 15 min at room temperature, and washed twice with PBS, 2×5 min. The migration ability of cells was evaluated by counting the number of invasive cells on the bottom of the inserts with a light microscope. The assay was performed in triplicate and was repeated at least three times.

Cell proliferation and invasion detected with the xCELLigence™ viability assay
The x CELLigence® RTCA DP system (Roche Applied Science, Penzberg, Germany) was used to automatically and continuously monitor live cell proliferation, growth, viability, migration, and spreading with characters of non-invasive, close to physiological status, a higher degree of accuracy, and lab free [29,30]. The cells transfected with siRNA-Control or si-BRWD3 were seeded on an E-plate-16 (ACEA Biosciences, San Diego, CA) at the optimal cell density (4×10 4 cells) with 100 µl DMEM media without FBS for proliferation assay. Cells' growth was recorded in real-time every 30 min. The cell growth status was exhibited with normalized cell index values. The assays were repeated three times.
As previously reported [29], the mixture of serum-free media and matrigel (Corning, NY, USA) was put into the upper chamber of CIM (cellular invasion/migration)-Plate 16. After 4 h, the cells transfected with siRNA-Control or si-BRWD3, 4×104 cells of MCF-7 or MDA-MB-231 were digested and re-suspended in serum-free DMEM and seeded into the insert chamber with FBS-free DMEM. The bottom chamber contained 165 µl of DMEM plus 10% FBS. The x CELLigence® RTCA DP system was used to monitor cell migration in real-time every 30 min. The difference between siRNA-Control and si-BRWD3 was shown with a normalized cell index. All data (including from the growth monitor assay) were expressed as mean±SD. Statistical analysis was performed with two-way ANOVA. Statistically signi cant was set as p<0.05.

Construction of plasmids and Cell transfection
No antibody was available for ChIP or even for immunoprecipitation for BRWD3, thus ChIP assay was performed with anti-GFP antibody (#AG281, Beyotime Biotechnology), which had been used in ChIP or immunoprecipitation assays [31,32]. The EGFP from pEGFP-N1 (Clontech) was ampli ed with PCR, and then the amplicon was inserted into the pcDNA3.1 (+) vector (Invitrogen, Thermo Fisher Scienti c) to construct pcDNA3.1-EGFP. Because of the large molecular weight of BRWD3 (203.5KDa), it's very di cult to introduce the BRWD3 into cell lines. So the sequences (3691-5815 bp) encoding two BRD repeats of BRWD3 was ampli ed with PCR and inserted into pcDNA3.1-EGFP to construct pcDNA3.1-2BRD (BRWD3)-EGFP. The primers and reaction conditions were all listed in supplementary table 1. Both the plasmid sequences were con rmed with the Sanger sequencing. Both pcDNA3.1-EGFP and pcDNA3.1-2BRD (BRWD3)-EGFP were introduced into MDA-MB-231 cells when the cell was grown to 50-60% con uence respectively. Transfection was con rmed by the assays of cell immuno uorescence and western blot. The plasmid of pcDNA3.1-2BRD (BRWD3)-EGFP was used as bait to immunoprecipitate speci c DNA fragments binding to BRWD3 through 2BRD, and the pcDNA3.1-EGFP was used as the matching control.
ChIP assay The ChIP was carried out referenced to a previous report [33]. Brie y, when the con uence of the cell transfected with target plasmids reached 80-90%, the cells were incubated with 1% formaldehyde in 1×PBS ( nal concentration of 0.75%) at room temperature for 10 min to chemically cross-link DNA to 2BRD(BRWD3). Glycine ( nal concentration of 125 mM) was added to quench the cross-link reaction.
Both immunoprecipitated DNA and input DNA were puri ed with the QIAquick® PCR puri cation kit (#28104, QIAGEN, Germany). The DNA concentration was measured with QUBIT dsDNA HS ASSAY KIT (Q32851, ThermoFisher, USA), and total DNA is required to be more than 10 ng.
HiSeq 2500 Illumina sequencing All samples were performed a conventional procedure, including end repair, adaptor ligation, size selection, ampli cation with PCR, enriched with AMPure XP beads, to construct library expressing 2BRD (BRWD3)-EGFP and control. The quality of libraries was evaluated with a 2100 Bioanalyzer High Sensitivity DNA chip, and the quantity was evaluated with the KAPA kit (# KK4602, illumina). Finally, the samples were subjected to HiSeq 2500 Illumina sequencing in CapitalBio Technology (China).
The quality of the raw data was checked with the FastQC software. Clean data was matched to the human genome (Ensemble GRCh38.81) with the bowtie1 [34]. Peak calling of unique mapped reads was conducted with MACS14 [35]. Annotation of ChIP peak, comparison, and visualization were conducted with ChIPseeker [36]. All the sites binding to pcDNA3.1-2BRD (BRWD3)-EGFP were selected through eliminating the sites binding to pcDNA3.1-EGFP. All sequencing data including will be deposited in NCBI's Gene Expression Omnibus when the article is exhibited.
Annotation and enrichment of gene functions All Ensemble gene IDs of sites were converted into gene symbols, then performed gene function annotation and enrichment by utilizing the Gene Ontology [37]. Availability of data and materials All sequencing data including will be deposited in NCBI's Gene Expression Omnibus when the article is exhibited. The relative method are available when in need.

Competing interests
No con ict of interesting.

Funding
The assay of yeast two-hybrid, cell culture and cell treatment, cell motility, and cell immuno uorescence was supported by the Natural Science Foundation of China (No.31371327) .

Supplementary Files
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