ATP‐dependent chromatin remodeller brahma related gene 1 promotes keratinocyte migration and modulates cell Signalling during wound healing in human skin

Skin wound healing is driven by proliferation, migration and differentiation of several cell types that are controlled by the alterations in the gene expression programmes. Brahma Gene 1 (BRG1) (also known as SMARCA4) is a core ATPase in the BRG1 Associated Factors (BAF) ATP‐dependent chromatin remodelling complexes that alter DNA‐histone interaction in chromatin at the specific gene regulatory elements resulting in increase or decrease of the target gene transcription. Using siRNA mediated suppression of BRG1 during wound healing in a human ex vivo and in vitro (scratch assay) models, we demonstrated that BRG1 is essential for efficient skin wound healing by promoting epidermal keratinocytes migration, but not their proliferation or survival. BRG1 controls changes in the expression of genes associated with gene transcription, response to wounding, cell migration and cell signalling. Altogether, our data revealed that BRG1 play positive role in skin repair by promoting keratinocyte migration and impacting the genes expression programmes associated with cell migration and cellular signalling.

5][16] These complexes consist of either BRG1 also known as SMARCA4 or Brahma (BRM) also known as SMARCA2 core ATPase in addition to other subunits. 15,16G1 controls terminal epidermal keratinocyte differentiation in mice 17,18 and humans. 14,19Moreover, it is essential for hair follicle epithelial stem cells maintenance, as well as activation and proliferation during induction of hair cycle and wound healing in mice. 20However, the potential functions of BRG1 in human epidermal keratinocytes during skin wound healing remained unknown.

| Research questions
To identify the role of BRG1 in control of human epidermal keratinocyte behaviour and alterations in gene expression programme during skin wound healing.

| Experimental design
See the supplementary experimental design file.

| BRG1 expression is upregulated in the wounded epithelium and promotes healing of human skin ex vivo
To identify a potential role of BRG1 in the early and intermediate stages of the skin wound healing process we employed the human ex vivo partial thickness excision wound healing model. 21We detected significant upregulation of the BRG1 immunofluorescence in the proliferating and migrating epithelial tongue in comparison to the distal epidermis on Day 3 post wounding (Figure 1A; Figure S1A), suggesting that BRG1 could be involved in the wound-induced re-epithelialization.
We transfected the wounds with the non-targeting control or BRG1 siRNA (siBRG1) (Accel SMARTpool-Human, Dharmacon) (Experimental design) and confirmed effective BRG1 silencing by significantly reduced BRG1 immunofluorescence in the wounded epithelium on Day 3 post-transfection (Figure 1B; Figure S1B).siBRG1 caused significant reduction of the wound edge length, yet, significant increase of edge thickness on Days 3 and 5 postwounding compared to the control (Figure 1C-F).A quantitative analysis of proliferation (Ki67) and apoptosis (active caspase 3) in the wound epithelium did not reveal any changes (Figure S1C-F).
However, the expression of stress-associated cytokeratin K16 and K17 was significantly reduced in the epithelial tongue of siBRG1treated wounds (Figure 1G,H; Figure S1G).These data indicate that BRG1 is involved in wound re-epithelialisation by promoting the keratinocyte migration associated with the upregulation of K16 and K17 expression. 22

| BRG1 gene suppression inhibits the human epidermal keratinocyte migration during the scratch-induced wound closure in vitro
To further ascertain the BRG1 involvement in wound-induced keratinocyte migration, we used an in vitro scratch assay. 23Cultured human primary epidermal keratinocytes (HPEK) were transfected with BRG1 or control siRNA for 48 h, then pre-treated with mitomycin C to inhibit proliferation, followed by the scratch (Figure S2A,B).siBRG1 caused significant inhibition of HPEK migration at 48 h after the scratch compared to the control (Figure 1I,J).K16 and K17 mRNA levels were markedly increased in the control HPEK, and in contrast, decreased in the siBRG1 transfected cells in 48 h after scratch (Figure 1K, L).Interestingly, siBRG1 did not have any effects on proliferation and apoptosis in intact (unstimulated by scratch) HPEK (Figure S2C-F).These data further confirmed a BRG1 involvement in keratinocyte migration required for proper wound re-epithelialisation.  of HPEK transfected with the siBRG1 versus control at 24 h after the scratch.In the control group, the significant changes (1.8-fold or higher) in the expression of 4634 genes were seen in response to the scratch.Of these, 1950 genes were upregulated (Table S1), and 2684 genes were downregulated (Table S2).The DAVID gene anthology (GO) analysis. 24of these gene sets identified several terms associated with the control of gene expression, wound healing and cell signalling.GO terms of positive regulation of cell migration, chemotaxis, inflammatory response, intracellular signal transduction, response to wounding, cell signalling, positive regulation of epithelial to mesenchymal transition, transcription initiation from RNA polymerase II promoter and regulation of cell adhesion among others were enriched in the upregulated gene set (Figure S3A).
GO terms of cell-cell adhesion, membrane organization, epidermis development and negative regulation of transcription among others were enriched in the downregulated gene set (Figure S3B).These data are consistent with previously published transcriptome changes in the epidermal keratinocytes during wound healing. 23,25 identify the BRG1-dependent genes that are induced by the scratch, the dataset of upregulated genes in the control HPEK was merged with the dataset of downregulated genes by siBRG1 at 24 h after the scratch.This analysis revealed 746 genes (about 38%) that are regulated by BRG1 (Figure 2A; Table S3).The DAVID GO analysis of these genes identified significant enrichment for biological terms associated with wound healing (Figure 2C), cell signalling (Figure 2C; The RT-qPCR analysis confirmed that siBRG1 downregulates the expression of the selected genes encoding signalling molecules CXCL3 and WNT4, chemokine receptor CXCR2, metalloprotease MMP7, protease inhibitor SERPINA3 and cytokeratin KRT80 in the scratch-stimulated HPEK (Figure 2D).Protein-protein interaction analysis performed using the STRING software tool (http:// strin g-db. org, vision 11.0) revealed the complex networks with several multiple connection nodes of the proteins encoded by the BRG1-induced up-regulated genes 26 (Figure 2E).Such nodes contained well-known regulators of wound healing, including TNFα, transcription factors FOS, SOX2 and EGR1.The nodes also include MAP2K6 kinase, heat shock proteins HSPA1A and HSPA1B, cell mobility-associated actins ACTG2 and ACTA1 and cyclin-dependent kinase CDK1 (Figure 2E).
Thus, our analysis of BRG1 mediated changes in the HPEK transcriptome during scratch wound healing revealed that BRG1 is involved in upregulation of many genes involved in wound healing, control of gene transcription, cell migration and cell signalling during wound healing.Such genes included TNF and genes encoding TNF pathway associated proteins.TNFα expression is upregulated in several cell types including keratinocytes upon skin wounding. 27TNFadeficient mouse keratinocytes have reduced cell migration capacity without change in their growth rate ex vivo and skin wound healing is significantly delayed in Tnf −/− mice. 28Moreover, TNFα treatment stimulates epithelial-mesenchymal transition in HEPK ex vivo. 29Our finding showing that reduced TNFα expression in the BRG1 deficient keratinocytes contributes to the decelerated keratinocyte migration and wound healing upon BRG1 gene suppression is consistent with these reports.
We also identified FOS protein as a one of key notes in interaction network of the proteins encoded by the BRG1 mediated upregulated genes.FOS gene expression is upregulated in different cell types, including keratinocytes, soon after wounding. 30It encodes an essential component of the activator protein-1 (AP1) transcription factor stimulating several different pathways, including metalloprotease expression 30,31 involved in keratinocyte migration during skin wound re-epithelialisation. 10 Inhibition of AP1 transcription factor leads to dramatically reduced HaCaT keratinocyte migration ex vivo. 31TNFα treatment leads to upregulation of the FOS expression in epidermal keratinocytes, 32 supporting an important link between TNF and FOS nodes in the interaction network of proteins that upregulation is mediated by BRG1 in HPEK during wound healing.To identify the BRG1-dependent genes that are suppressed by the scratch, the dataset of downregulated genes in the control HPEK was merged with the dataset of upregulated genes in the siBRG1 at 24 h after the scratch revealing 299 genes (about 11.14%) (Figure 2B; Table S4).The DAVID GO and STRING analyses suggest that BRG1 is required for downregulation of genes involved in RNA processing, mRNA splicing, ribosomal biogenesis and protein translation during wound healing (Figure 3).

| CON CLUS I ON S AND PER S PEC TIVE S
Here we demonstrated that BRG1 promotes wound re-

ACK N OWLED G EM ENTS
This work was funded by BBSRC Industrial CASE CVS BB/ M50273X/1 and Unilever to Michael Fessing.

CO N FLI C T O F I NTE R E S T S TATE M E NT
This work was partially funded by Unilever.Ranjit Bhogal is an employee of Unilever.We do not claim any other conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Datasets related to this article have been submitted to and are available at https:// www.ebi.ac.uk/ biost udies/ studi es/ E-MTAB-12452 .

F I G U R E 1
BRG1 is required for the wound healing in human skin ex vivo and in vitro by promoting keratinocyte migration.(A) BRG1immunofluorescence in the ex vivo partial thickness wounds: prominent and low BRG1 expression in the wound epithelial tongue and the adjacent unwounded epidermis, respectively, on Day 3 post wounding.Scale bars 100 μm.(B) BRG1 immunofluorescence in the wound epithelial tongue transfected with siBRG1 versus and control siRNA on Day 3 post wounding.Scale bars 100 μm.(C,D) Haematoxylin staining of the control siRNA or siBRG1 treated skin on Days 3 and 5 after wounding.Dotted lines indicate the wound epithelia.Scale bars 100 μm.(E) Measurements of the epithelial tongue length in the control or BRG1 siRNA treated wounds on Days 3 and 5 post-wounding.n = 3 donors per a treatment/time point, 5 measurements per a sample, all data points and means are shown, ***p < 0,001, Student's t-test.(F) Measurements of the epithelial tongue thickness in the control or BRG1 siRNA treated wounds on Days 3 and 5 post-wounding.n = 3 donors per a treatment/time point, 5 measurements per a sample, all data points and means are shown, ***p < 0,001, Student's t-test.(G, H) Decreased K16 (G) and K17 (H) immunofluorescence in the siBRG1 treated vs the control siRNA on Day 3 after wounding.Scale bars 100 μm.(I) Representative images of the HPEK transfected with the control or siBRG1 at 24 and 48 h after scratch.Black hatch lines outline the wound edge in 1 h after the scratch; the red hatch line indicates the wound edge at the specified observation time.Scale bars 500 μm.(J) Wound width at 1, 24 and 48 h after the scratch (n = 3 wells per a group, 20 measurements per a well.All data points and means are shown, ***p < 0.001, Mann-Whitney U-test).(K, L) RT-qPCR analysis of the K16 (K) and K17 (L) mRNA expression in siBRG1 and control transfected HPEK at 24 and 48 h after the scratch normalized to GAPDH (n = 3 donors; data points and means are shown; *p < 0.05, Student's t-test).

2 . 3 |
BRG1 controls changes in gene expression programme associated with the epidermal keratinocyte migration, cell-signalling, gene transcription regulation, RNA processing, ribosomal biogenesis and protein translation during skin wound healing in vitroTo define the BRG1-dependent changes in the gene expression in the HPEK, we performed microarray transcriptome profiling (Agilent, SurePrint G3 Human Gene Expression v3 8 × 60K Microarrays)

Figure
Figure S4A), gene transcription (Figure 2C; Figure S4B), and immune response/defence against pathological microorganisms and viruses (Figure 2C; Figure S4C).The enriched wound healing terms included chemotaxis, positive regulation of neutrophil chemotaxis, inflammatory response, cell migration, angiogenesis and regulation of cell-cell adhesion among others (Figure 2C).Enriched signalling pathway components included nitric oxide mediated signal transduction, canonical Wnt signalling pathway, positive regulation

F I G U R E 2
BRG1-mediated up-regulation of gene expression in the HPEK at 24 versus 1 h after the scratch-induced wounding.(A) Vann diagram indicating number of different and shared genes that are upregulated in the control versus downregulated genes in siBRG1 transfected HPEK at 24 h after the scratch.(B) Vann diagram indicating number of different and shared genes that are downregulated in the control versus upregulated genes in siBRG1 transfected HPEK at 24 h after the scratch.(C) GO DAVID enrichment analysis showing significant over-representation for wound healing associated biological processes of the 746 genes upregulation of which is suppressed by siBRG1 in HPEK in response to the scratch.(D) RT-qPCR analysis of the selected gene expression in the siBRG1 transfected HPEK versus the control at 24 h after the scratch (set as value 1) normalized to GAPDH (n = 3 donors, datapoints and means are shown; *p < 0.05, **p < 0.01, ***p < 0.001, ns, non significant, Student's t-test).(E) The STRING protein-protein interaction network analysis showing the association between proteins encoded by the genes upregulation of which is suppressed by siBRG1 in HPEK in response to the scratch.
epithelialisation in human skin by controlling (i) cell migration; (ii) induction of genes associated with cell migration, cell signalling, cellular response to stress and control of gene transcription; and (iii) suppression of genes associated with RNA modifications and splicing, control of ribosome biogenesis and protein translation.Our report provides the foundation for identification of gene promoters and distal gene regulatory elements directly controlled by BRG1 in human epidermal keratinocytes during wound healing and how the defects in control of this gene regulatory elements could lead to wound healing abnormalities including chronic wounds.This new insight in the mechanisms controlling epidermal keratinocyte activation, cell migration and cell signalling will hopefully result in future development of new treatment approaches for chronic wounds targeting defects in BRG1 mediated mechanisms of gene regulation and the molecular pathways controlled by the BRG1 target genes.AUTH O R CO NTR I B UTI O N S Conceptualization: NVB, MYF; Data curation: CK, MYF; Formal analysis: CK, MYF; Funding acquisition: RKB, MYF; Investigation: CK, RKB, NVB, MYF; Methodology: CK, MYF; Project administration: RKB, MYF; Resources; RKB, MYF; Software: CK, MYF; Supervision: NVB, RKB, MYF; Validation: CK; Writing-Original draft preparation: CK, NVB, MYF; Writing-review and editing: CK, RKB, NVB, MYF.All authors read and approved the final manuscript version.
Kellett https://orcid.org/0000-0002-0753-190XRanjit K. Bhogal https://orcid.org/0000-0003-4835-4871F I G U R E 3 BRG1-mediated downregulation of gene expression in the HPEK at 24 versus 1 h after the scratch-induced wounding.(A) GO DAVID enrichment analysis showing significant over-represented biological processes for the 299 genes downregulation of which is suppressed by siBRG1 in HPEK in response to the scratch.(B) The STRING proteinprotein interaction network analysis showing the association between proteins encoded by the genes downregulation of which depends on BRG1 in HPEK in response to the scratch.