Transcription Factor Binding Site Analysis Identifies FOXO Transcription Factors as Regulators of the Cutaneous Wound Healing Process

Karl Markus Roupé,Stephen M Hedrick,Erica L Stone,Srinivas Veerla,Victor Nizet,Ole E Sørensen,Joshua Olson,Andrea Motta

doi:10.1371/journal.pone.0089274

Karl Markus Roupé, Stephen M Hedrick + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0089274

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Journal: PLoS ONE	Publication Date: Feb 19, 2014
Citations: 91	License type: CC BY 4.0

Affiliation: University of California, San Diego, Lund University

Abstract

The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation.

Highlights

It is increasingly recognized that stable clusters of co-occurring transcription factor binding sites (TFBS) coordinately regulate gene sets associated with highly specific cellular activities [1–3]
We investigated the presences of co-occuring TFBS for FOXO1, FOXO4 and FOXO3 and detected all three Forkhead box ‘‘O’’ (FOXO) factors within 50 base pairs of each other in the majority (71%) of the promoter regions of the 98 genes (Figure 1A, 1B)
Our finding that the absence of FOXO3 is beneficial for the wound healing process in mice in vivo could possibly contribute to explaining some of the pathophysiology of impaired wound healing seen in the elderly

Summary

Introduction

It is increasingly recognized that stable clusters of co-occurring transcription factor binding sites (TFBS) coordinately regulate gene sets associated with highly specific cellular activities [1–3]. We hypothesized that a search for significant enrichment of TFBS, located in close proximity to one another in the promoter regions of the most differentially expressed genes in genome wide microarray data set, would represent a powerful approach to find key regulators of a complex biological process. We tested this hypothesis by re-analyzing two published data sets on the human epidermal response to injury using the TFBS analysis program, Systematic Motif Analysis Retrieval Tool (SMART). The combined presence of resident dermal cells and infiltrating inflammatory cells in some of the studies have made it difficult to delineate important pathways and attribute specific roles to keratinocytes

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