Background: Transcriptional mechanisms that regulate epidermal homeostasis have been well established but recently we have discovered that post-transcriptional mechanisms play prominent roles in maintaining epidermal self-renewal. We have shown that the RNA helicase DDX6 is necessary to maintain epidermal self-renewal through the mRNA degradation and translation pathway. By associating with specific members of the translation pathway DDX6 binds to and mediates the translation of self- renewal and proliferation transcripts to maintain self-renewal. DDX6 also associates with mRNA degradation proteins to bind differentiation-inducing transcripts to promote their degradation to prevent premature differentiation. Objective/hypothesis: This proposal seeks to understand the regulation of epidermal stem and progenitor cell self-renewal and differentiation through post- transcriptional mechanisms. We previously identified proteins associated with DDX6 by mass spectrometry and our objective is to characterize the role of each protein in regulating epidermal growth and differentiation as well as the mechanisms of action. Furthermore we seek to determine the specific transcripts that DDX6 and its associated complexes bind.
Specific Aims : (1) To determine the role of DDX6 associated proteins on epidermal homeostasis and (2) to identify and characterize the transcripts associated with DDX6 complexes. Study Design: To study epidermal homeostasis in a more clinically relevant setting, we generate 3-dimensionally intact human skin, containing human epidermal cells (that have been permanently knocked down for either DDX6 or its associated proteins) in the context of human dermal stroma and basement membrane, regenerated on immune compromised mice. By using this model, we can perform loss of function experiments on DDX6 and its associated proteins in regenerated human skin to characterize their role in epidermal growth and differentiation. We will also use RNA immunoprecipitations followed by next generation sequencing to determine the RNAs associated with DDX6 complexes.
Alterations in the growth and differentiation of epidermal stem and progenitor cells can lead to a variety of disorders including non-melanoma skin cancers as well as chronic wounds. Thus a comprehensive understanding of the mechanisms that regulate epidermal growth and differentiation is necessary to understand the progression to disease. This proposal seeks to characterize the mechanisms of epidermal self-renewal and differentiation through post-transcriptional regulation.
