Project description:How cell and tissue identity persist despite constant cell turnover is an important biologic question with cell therapy implications. While many mechanisms exist, we investigated the controls for site-specific gene expression in skin given its diverse structures and functions. For example, the transcriptome of in vivo palmoplantar (i.e. volar) epidermis is globally unique including Keratin 9 (KRT9). While volar fibroblasts have the capacity to induce KRT9 in non-volar keratinocytes, we demonstrate here that volar keratinocytes continue to express KRT9 in vitro solo-cultures. Despite this, KRT9 expression is lost with volar keratinocyte passaging, in spite of stable hypo-methylation of its promoter. Coincident with KRT9 loss is a gain of the primitive Keratin 7 and a signature of dsRNA sensing, including the dsRNA receptor DDX58. Exogenous dsRNA inhibits KRT9 expression in early passage volar keratinocytes or in vivo footpads of wild-type mice. Loss of DDX58 in passaged volar keratinocytes rescues KRT9 and inhibits KRT7 expression. Additionally, DDX58 null mice are resistant to the ability of dsRNA to inhibit KRT9 expression. These results demonstrate that the sensing of dsRNA is critical for loss of cell specific gene expression; our results have important implications of how dsRNA sensing is important outside of immune pathways. Keratinocytes were expanded from both the sole and the dorsum of the foot and at passage 4, RNA was extracted and sent for microarray analysis
Project description:How cell and tissue identity persist despite constant cell turnover is an important biologic question with cell therapy implications. While many mechanisms exist, we investigated the controls for site-specific gene expression in skin given its diverse structures and functions. For example, the transcriptome of in vivo palmoplantar (i.e. volar) epidermis is globally unique including Keratin 9 (KRT9). While volar fibroblasts have the capacity to induce KRT9 in non-volar keratinocytes, we demonstrate here that volar keratinocytes continue to express KRT9 in vitro solo-cultures. Despite this, KRT9 expression is lost with volar keratinocyte passaging, in spite of stable hypo-methylation of its promoter. Coincident with KRT9 loss is a gain of the primitive Keratin 7 and a signature of dsRNA sensing, including the dsRNA receptor DDX58. Exogenous dsRNA inhibits KRT9 expression in early passage volar keratinocytes or in vivo footpads of wild-type mice. Loss of DDX58 in passaged volar keratinocytes rescues KRT9 and inhibits KRT7 expression. Additionally, DDX58 null mice are resistant to the ability of dsRNA to inhibit KRT9 expression. These results demonstrate that the sensing of dsRNA is critical for loss of cell specific gene expression; our results have important implications of how dsRNA sensing is important outside of immune pathways. Keratinocytes were expanded from the sole of the foot and at passage 4 or 8, RNA was harvested to identify unique transcripts
Project description:Human epidermal keratinocytes undergo tightly controlled program of cell differentiation, leading to the formation of cornified envelope. Primary keratinocytes in vitro, under calcium stimulation mimic the differentiation program observed in vivo. Analysis of the transcription profile of two cell population, such as proliferating cells and differentiating cells helps to discover new genes implicated in that process and to understand the mechanisms of regulation of the keratinocyte differentiation. Primary human keratinocytes were cultured under proliferating (Day 0, sub-confluent cells) and differentiating (seven days of high calcium medium) conditions. As a source of cells, we used normal skin from different body sites: back, foreskin, palmoplantar. RNA extracted from cultured primary human keratinocytes were isolated from five different donors. We compared the expression profiles of proliferating versus differentiating keratinocytes.
Project description:Human epidermal keratinocytes undergo tightly controlled program of cell differentiation, leading to the formation of cornified envelope. Primary keratinocytes in vitro, under calcium stimulation mimic the differentiation program observed in vivo. Analysis of the transcription profile of two cell population, such as proliferating cells and differentiating cells helps to discover new genes implicated in that process and to understand the mechanisms of regulation of the keratinocyte differentiation.