Formation of skin barriers by epigenetic regulation of EGR3. Kim Kyu-han

Skin Barrier Control Factor

Skin barriers play a very important role in protecting the skin and various body organs in the skin from various harmful factors in the external environment. In the base layer of the skin's epidermis, keratin-forming cells divide, and as they go through the process of differentiation, they move to the upper layer of the epidermis. The differentiated keratin-forming cells in the granulate layer of the epidermis begin to express filaggrin, involucrin, and loricrin, which are important proteins for skin barrier formation. Eventually, it forms a keratin in the epidermal keratin layer [1].

In this formulation, keratin-forming cells undergo very large genetic changes with numerous gene expression changes. In the meantime, there have been relatively many studies on genetic changes in this differentiation process. However, relatively few studies have found on selective genetic modifications in the granule layer, a key part of forming skin barriers. Unlike the keratin layer, the granulate layer consists of living cells and from this layer, the expression of genes such as pilaggrin, involucrin, and lorikrin, which can be called skin barrier forming markers, begins to maximize. Little was known, however, about the regulatory factors specific to this granulation layer.

There have been studies of transcriptional factors known to be important for epidermal differentiation, but since most of their expression begins below the granulate layer, It has been insufficient to call them specific regulatory factors for skin barrier formation.

Therefore, the study of transcriptional factors that exhibit a specific manifestation in the granulated layer, which is crucial to the formation of skin barriers was begun because It was thought that finding such transcription factors would not only reveal the secret details of the skin barrier, but also help develop cosmetics that protect the skin barrier.

Discovery of EGR3

Various bio big data have been utilized to conduct research to find transcriptional factors that exhibit particular expressions in the granule layer [2]. Such big data was produced on its own, but it also actively utilized data used in several bio big data-related databases and papers already published. First, the RNA profile data generated from the keratin differentiation model performed in an inbitro manner selected genes that underwent a differentiation process similar to the granule layer. Approximately 1,000 genes were selected, of which only the transcription factors were selected in the next phase. And the third step was to explore the skin expression patterns of candidate genes using a database of data dyed by immunosytochemical methods in skin tissue.

Eventually, we found that Early Growth Response Protein 3(EGR 3) was a specific tran-scriptional factor in the epidermal granules, which was not known at all about the role it has played in the skin, and thus began to focus on its skin function and mechanism.

EGR3 Skin Barrier Formation Function

Prior to conducting an experiment on the skin barrier formation function of EGR3, in order to determine whether EGR3 is related to skin barrier within bio-big data provided in various open source forms, in-silico analysis was performed first.

First, we looked at the expression of EGR3 in various skin diseases, and observed that the expression of EGR3 decreased significantly in atopic and skin squamous cell carcinomas, which are known to have broken skin barriers.

Further confirmation of EGR3's expression in various skin disease samples showed rapid reduction in EGR3's expression in samples showing non-degradable cell nuclei in keratin layers due to poor epidermal differentiation. In other words, EGR3 could only be concluded to be selectively expressed in the granules of the epidermis during normal skin development.

We then infer the capabilities of EGR3 by leveraging the GTEx database (https://www.gtexportal.org/home/)),

which provides RNA expression profiles in various human tissues. After dividing skin tissue samples in the GTEx database into groups with high EGR3 and low EGR3 expression groups, analysis of RNA expression profiles in both groups in bioinformatics showed that there was much more expression of epidermal differentiation and skin barriers in groups with high EGR3. This is an indirect indication that EGR3 is associated with skin barriers.

We experimentally confirmed whether EGR3's skin barrier formation function, which we learned through in-silico analysis, is really correct. After performing a functional loss experiment that inhibited EGR3 with siRNA, we examined the RNA profile through RNA-sequencing (RNAseq) and observed that without EGR3, the expression of various genes associated with skin barriers is significantly reduced. Furthermore, we produced and examined artificial superficial cells without EGR3 and observed that without EGR3, it shows negative keratosis and significantly reduces the expression of pilaggrin, a skin barrier marker protein. This shows that EGR3 plays a very important role in skin barriers.

Epigenetic Regulator of EGR3

When analyzing the data using a biometric analysis method, it was found that EGR3 was closely related to the Chr1q21 part, a specific part of the human genome No. 1. In fact, this site is well known as the Epidermal Differentiation Complex, where various genes, including pilaggrin, involucrin, and lorikrin, are important in forming skin barriers are forming clusters. RNA-seq data from samples that reduced the expression of EGR3 with siRNA showed that without EGR3, the expression of most genes located in the skin differentiation gene complex was significantly reduced.

Furthermore, various epigenetic methods and tools have been used to find out how EGR3 integrally regulates skin differentiation gene complexes. First, we performed ChIP-sequencing, a technique that tracks the area where EGR3 sticks directly to the dielectric, and looked at what area EGR3 sticks to in the skin differentiation gene complex, and observed that EGR3 sticks to three major areas. Furthermore, various histone proteins have been investigated to show that the site to which EGR3 sticks has the form of an enhancer known to regulate the expression of other sites in DNA. In other words, EGR3 was identified to perform integrate control of the expression of a number of skin barrier genes through enhancers in the skin differentiation gene complex.

Next, we looked at how EGR3 controls the enhancer. We first conducted an enhancer-luciferase experiment to see if EGR3 regulates the activity of the three regions that bind directly, and confirmed that EGR3 regulates the activity of the amplifier in two areas. However, the EGR3 binding site present near the lorikrin gene did not increase its activity by EGR3.

Recent research has shown that non-coding RNA, which does not form proteins in the enhancer site but plays a very important role, has functioned as enhancers[3]. The non-coding RNA expressed in these enhancers is called the enhancer RNA

Interestingly, we found that RNA polymerases known to express RNA in EGR3 binding sites in the lorikrin site were bound, and we thought that this might lead to expression of non-coding RNA in these sites. A PCR method was used to confirm that when keratin-forming cells are differentiated, rapid RNA is expressed in this area. It was also confirmed that artificially inhibiting this expression significantly reduces the expression of various genes in the skin differentiation gene complex. In other words, EGR3 integrally regulates the skin differentiation gene complex, which uses a two postgenetic methods; increasing the activities of the enhancer and expressing the enhancer RNA.

EGR3 Utilization Study: Skin Barrier Enhancement Function of Peony Root Extract

Peony root extract is known to have several medicinal properties, including anti-inflammatory, and is also used as a raw material for cosmetics. However, the function of peony roots associated with skin barriers is not well known. RNA-seq experiments have shown that peony roots increase expressions such as pilaggrin, involucrin, and loricrin, which strengthen skin barriers [4]. The UHPLC method also showed that the main components of peony roots were paeoniflorin, albiflorin, and PGG,

among which PGG increased the expression of pilaggrin, involucrin, and lorikrin. This shows that the PGG of peony root extract has a function to strengthen skin barrier formation.

Furthermore, we examined various transcription factors from RNA-seq data that was treated by peony root extracts and confirmed that only EGR3 is selectively increased. Further confirmation of this with the RT-qPCR method also confirmed that EGR3 is increased by PGG.

Conclusion

EGR3 is a regulating factor that is only expressed in the granulate layer of the skin's epidermis, which does not occur properly in skin diseases where skin differentiation does not take place. EGR3 has been shown to play a very important role in skin barriers through various bioinformal analysis and artificial skin experiments.

In addition, the epidermal differentiated gene complex, which contains a number of genes essential to the formation of various skin barriers, is synthetically controled, using epigenetic methods such as activation of enhancers and promoting the expression of amplifiers RNA.

PGG, one of the key components of peony root extract, promotes the expression of skin barrier genes in skin keratin-forming cells through the promotion of EGR3. Therefore, peony root extract is thought to be useful as a cosmetic material as it helps strengthen skin barriers.