ABSTRACT

Wound healing is a highly complex mechanism involving overlapping stages which starts with a phase of inflammation followed by proliferation and reepithelization. The overall effect is to remodel the injured skin back to normal through remodelling.

The SWI/SNF complex is involved in remodelling histone tails allowing nucleosome remodelling .This complex is composed Brm or Brg1 along with BAF complex that aid in expression of genes.

Our Research was conducted on the analysis of Brg1 in keratinocytes during wound healing.

This was conducted using an Immunohistochemistry approach using 4 skin tissues from mice  including an 8 week Telogen tissue used for a point of comparison. Further three tissue showed progressive wound healing from day 3, 5 and 7.

Analysation of Fluorescent intensity was conducted quantitatively. Results showed a progressive increase expression of Brg1 from the distal to distal hyperproliferative regions from day 3 to day 7.

The change Brg1 expression suggests that Brg1 plays a role in the expression of genes involved in proliferation and differentiation of keratinocytes,

High intensity of Brg1 In the epithelial tongue was detected suggesting Brg1 plays a role in expressing the integrin genes allowing migration over the denuded area of the wound.

Qualitative visual analysis showed higher fluorescent intensity at hair follicle from the distal to hyper proliferative of wounds suggesting that Brg1 plays a role in maintaining the stem cell pool of hair follicle

1- INTRODUCTION

1.1 Functions of the skin

The mammalian skin provides a permeable barrier against the loss of water from nucleated epidermis layers whilst maintaining a ph of 5.5 supressing microbial growth. The skin also protects against UV and external factors such as chemicals and pathogens. Receptors on the surface of the skin allow thermoregulation as well as sensing pressure touch and pain (Boer et al,2016).

1.2 Structure of the Skin

The epidermis is a stratified epithelium formed up of organised layers.  The keratinocytes are the most abundant cells which switch from a proliferative state in the basal layer to a differentiated state as they migrate through the suprabasal layer to form the stratum corneum. At the cornified layer the keratinocytes dehydrate and flatten forming corneocytes. This layer is resistant to abrasion forming a barrier against external factors whilst preventing water loss (Simpson et al 2011).

The intermediate filament Keratin form the infrastructure of keratinocytes as the cells move through the spinous and granular layer of the suprabasal layer undergoing differentiation there is a switch of keratins from K5 and K14 to K1 and k10.( Pastar et al 2014,moll et al 2008). The keratinocytes at the basal cells are attached to basement membrane through hemidesmosomes whilst at the suprabasal cells attach to neighbouring cells via desmosomes (Simpson et al 2011). Migration of keratinocytes during wound healing and maintenance of lost cells requires disconnection of attachments.

The process of differentiation requires MAP kinase pathways activated by various stimuli including a calcium influx and epidermal growth factors (Jost et al 2001 , Pastar et al 2014).

Further cells present in the epidermis include Melanocytes, Langerhans cells, Lymphocytes and Markel cells. These cells are involved in producing pigment and immunological defence respectively.

The extracellular matrix of the dermis composed of collagen; fibril and elastic fibres help provide structural support to the dermis. Fibroblasts cells are prominent in the dermis where they secrete collagen which forms a meshwork providing strength and elasticity. The network of capillaries and sweat glands are responsible for regulation of temperature through homeostasis (Tracy et al 2016).

The hypodermis is the last layer formed up of subcutaneous fat with a role in protection and insulation.

1.3 Wound healing

Injury to the skin involves cutaneous wound healing through overlapping processes. Immediately after a wound haemostasis begin with platelet aggregation leading to fibrin clot activation. Pro inflammatory cytokines are released from clot and surrounding tissue including Fibroblast, epidermal- Transforming and platelet derived growth factors. The role of neutrophils includes clearance of microbes and debris using lysosomal enzymes. The macrophages release cytokines which activate leukocytes and remove apoptotic cells allowing tissue regeneration. T cell lymphocytes maintain wound healing through tissue integrity maintenance by regulating inflammation (Guo et al, 2010).

During the inflammatory phase the proliferative phase begins characterized by the entry of fibroblasts and angiogenesis.  The fibroblast produce components of the extracellular matrix (ECM) which aid migration composed of collagen, fibronectin and glycol proteins. The blood vessels, cells and the ECM contribute to the granulation tissue at the wound bed which allows reepithelization of the wound to occur (Guo et al, 2010).

To close the defect in epidermis the keratinocytes at the wound edge loosen their adhesion to each other and to basal lamina via disassembly of desmosomes and hemidesmosomes. Keratinocytes behind the migrating tongue proliferative whilst at the wound edge they migrate over the denuded area. This occurs via activation of PKCa which lead to conversion of calcium independent to dependent desmosomes. Transcription factor Slug allows for release of keratinocytes.  Viscoelasticity is increased via the change of keratins as the keratinocyte migrate up the layer regulated by growth factors present within wound environment. (Pastar et al 2014)

This is followed by the remodelling phase where inflammatory cells go through apoptosis and collagen is replaced returning to normal skin.

1.4 Brg1 in embryonic development

Nanog is the transcription factor involved in self-renewal of undifferentiated embryonic stem cells (ESC) for pluripotency. Initially expressed widely during the morula-to-blastocyst transition, then expressed only in inner cell mass and silenced as the cells differentiate in the extraembryonic lineages (Moery et al 2015).  Brg1 acts as a repressor and activator of transcription of genes through interaction with additional proteins.

The initial regulation of Nanog occurs in the embryonic development where it is vital to maintain the pluripotent characteristics of these stem cells hence this involves an increase in Noggin.  Brg1 or its corepressor HDAC1 are disrupted leading to an increase in acyletayed H3K9/14 at the Nogginproximal enhancer allowing an open chromatin for expression.

Further development during the trophoblast lineage involves silencing of Noggin. Brg1 and HDAC1 are maintained the effect is acH3K9/14 are downregulated at Noggin proximal enhancer preventing an open chromatin structure.(Carey et al 2015)

Chromatin remodelling factors also interact with the tumour suppressor protein p53 to maintain the balance between proliferation and apoptosis. Chromatin transactivation protein BRG1 interact with p53 controlling the levels. Deletion of the brg1 in embryonic development results in peri-implantation lethality whilst an increase in Brg1 leads to low levels of p21 and p53 leading to rapid embryonic growth ( Marquez-Vilendrer et al 2016) (Singh et al 2016).

1.5 Brg1 and Hair follicles

Hair follicles contribute to epidermal healing through emigration of stem cells from follicle aiding in reepithelization. Absence of hair follicles results in a delay in wound closure and the epidermis hyper proliferate further away from site of wound.  The hair follicle is divided into three phases consisting of: Anagen (Growth phase), Catagen (regression phase) and Telogen (resting phase) Replenishment of cells involves the epidermal stem cells (ESC) niches. These include the bulge of the hair follicle, the base of sebaceous glands and the basal layer of the interfollicular epidermis (Ito et al,2008).

The stem cells in the basal layer are unipotent In comparison to the multipotent stem cells found in the bulge cells of the hair follicles which are vital in maintenance of the IFE stem cells. During the Telogen the HFSC are kept inactive by BMP and FgF18. Activation of the hair follicle bulge cell progeny move into the lower outer root sheath towards the matrix. Entry into anagen involves BMP inhibitors from dermal papilla of hair follicle (Blanpain et al 2006). Brg1 binds to the promoter of Sonic hedgehog genes upon stimulation SHH signal causes bulge HFSC to divide symmetrically causing the outer root sheath to grow which moves signalling center away at dermal papilla returning the bulge to quiescence. Brg1 is activated by skin injury to facilitate early epidermal repair. Studies demonstrate a molecular circuit that integrates chromatin remodelling (Brg1), transcription regulation (NF-KB, Gli) and intercellular signalling Shh to control bulge stem cells during tissue regeneration (Xiong et al, 2013).

1.6 Brg1 in epidermal development

Epidermal development during embryogenesis occurs at E9.5 followed by stratification at E14.5. At E15.5 the spinous layer is formed. Terminal differentiation of the epidermis occurs at E18.5 (Koster et al 2007).The epidermis of the embryo lacking Brg1 still enters stratification but the terminal differentiation of keratinocytes is affected. Brg1-null mice showed disorganisation at stratum corneum due to alteration of lipid composition and number of corneodesmosomes that hold the stratum corneum together. (Indra et al  2005)

The transcription factor p63 regulates epidermal development by regulating a large number of genes. The epidermal differentiation complexes (EDC) are a cluster of genes involved in terminal differentiation and cornification of Keratinocytes found on the spanning 1.6 Mb on human 1q21 and 3q chromosome in mice.

P63 regulates the expression of the A-T rich binding protein SATB1. This protein is involved in the higher order chromatin folding of keratinocyte. P63 deficient mice showed a downregulation of chromatin regulating factors including Brg1 (Fessing et al 2011).

Regulation of Brg1 was found to be vital during epidermal development via p63. Brg1 was found to relocate the EDC towards the nuclear interior enclosed   in SC35-positive speckles enriched in RNA metabolic factor which facilitated efficient transcription of genes (Mardarev et al 2014).

1.7 Aims of the project

The aim of the project is to analyse the expression of Brg1 in keratinocytes during cutaneous wound healing. Mice skin of progressive wound would be provided and using immunohistochemistry techniques to detect expression of the protein.

The project will allow a wider understanding of Brg1 in different stages of wound healing by looking at different areas of the wound including the distal, distal hyperproliferative and the epithelial tongue.

2.0 MATERIALS AND METHODS

 2.1 Immunofluorescent staining

Mouse skin cyrosections were used for the detection of Brg1/Baf155. Tissue sections of variable times point from day 3, 5 and 7 showing progressive wound healing were used for analysis.  A wild type 8 week Telogen tissue was used as a control for comparison.

The slides were removed from storage at -80°C and air dried for 10 minutes before being placed in 4% Formaldehyde for 10mins. This was followed by three consecutive washes with 1 x PBS for 5 mins (refer to table 1). The tissue sections were encircled with the PAP pen and dried for 2-3mins.

100µl of the blocking buffer (1% BSA/0.1%Triton x-100 / 0.05% Saponin in PBS) was added to each section. Incubation occurred at room temperature for 1 hour in a humidified chamber.

Optimum dilutions were investigated for Brg1 primary antibodies using the blocking buffer and at 8 week Telogen tissue sample. Brg1 santacruz was tested using 1:50, 1:100 and 1:200 dilutions. Brg1millipore and Santa cruz were tested using the same dilutions of 1:100, 1:200 and 1:400.

Optimum dilution for Baf155 was tested at 1:200 and 1:400. The preferred concentration of 1:400 was used using the blocking buffer produced. 100µl of these dilutions were placed on each tissue section. Overnight incubation in a moisturizing chamber occurred at 4°C.

Slides removed from incubation were washed with 1xPBS 3 times for 5 mins. Secondary antibody Donkey anti rabbit biotechnology was used with the recommended 1:200 dilution for 1 hour in a humidifying chamber in an incubator at 37°C by placing 100µl on each tissue section.

Goat anti Mouse IgG H&L (alex fluroa488) was used for Brg1 mouse primary antibody. The secondary antibody was diluted with1% BSA in 1x PBS.

Excess antibody was removed with 3 x washes with 1 x PBS for 5 mins. This was followed by fixing using vector labs vectashield medium with DAPI (abcam ab104139) and a coverslip (24-60mm) was placed on each slides. Nail varnish coat was applied on the edges of the coverslip.

2.2 Florescent and imaging slides

In order to visualise the expression of the proteins the slides were viewed and images captured using an epiflorescent microscope (Nikon ecilipse 80) and Image pro insight 64 Image j was used to analyse the images.  A 10x magnification was used to capture images of the full wounds.

Analyses required 20x magnifications using an exposure of 10 milliseconds for the detection of DAPI of each sample whilst an exposure of 300 milliseconds was used to view the secondary antibody. Three images were taken of the tissue section to allow valid comparison these included distal ends to show normal epithelium and distal hyper proliferative and epithelial tongue images to show progressive wound healing. Again images were merged using image j.

2.3 Semi quantitative analysis

Images were analysed using Microsoft excel along with image J. A comparison of protein expression was calculated using same area boxes by calculating integrated density of the tissue with florescence and a region of blank space of background. These steps were repeated for other areas in field.

Once completed results were copied into programmed excel sheet which provided results for comparison .The average of the background was subtracted from the mean antibody expression.  The results were then used to form a bar chart to show comparison between the distal and distal hyperproliferative regions and the epithelial tongue. This method was modified from a previous research project (McCloy et al 2014)

2.4 Statistical analysis.

The mean and the standard deviation of the blank space background and the expression of protein was calculated using the programmed excel sheet.

Table 1

Table -1