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:The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate – whether to remain proliferative or terminally differentiate – is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome-wide MethylationEPIC beadchip array measuring approximately 850,000 probes combined with RNA sequencing of in vitro cultured nondifferentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes and only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. TRIM29 knockdown resulted in significantly lower expression levels of terminal differentiation genes as a group, although differences for individual genes were considered minor (<2 fold). From these results we conclude that it is unlikely that DNA methylation has a main regulatory role in terminal keratinocyte differentiation regulation in vitro.
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.
Project description:Targets of Retinoic Acid (RA) and 3,4-didehydroretinoic acid (ddRA) were identified in primary human epidermal keratinocytes grown in the presence of atRA or ddRA for 4 and 24 hours. Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (more than 350) than in proliferating keratinocytes (20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids. Proliferating and differentiated keratinocytes were harvested after 4 and 24 hours after treatment with atRA, ddRA and vehicle for RNA extraction and hybridization on Affymetrix microarrays. Triplicate samples were generated for each time point and each treatment.
Project description:Targets of Retinoic Acid (RA) and 3,4-didehydroretinoic acid (ddRA) were identified in primary human epidermal keratinocytes grown in the presence of atRA or ddRA for 4 and 24 hours. Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (more than 350) than in proliferating keratinocytes (20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids.
Project description:This SuperSeries is composed of the following subset Series: GSE35379: Genome-wide occupancy map of GATA-1 in proliferating and differentiating murine ES cell derived erythroid progenitors (ES-EP) GSE35384: Transcriptome analysis of differentiating normal and leukemic erythroid progenitors Refer to individual Series
Project description:We find GATA-1 occupies 6,600 sites in proliferating erythroid progenitors and 10,600 sites in differentiating progenitors. 80-90% of GATA-1 binds within intragenic or intergenic regions, while <20% of GATA-1 is found within 2kb of TSS. Assaying GATA-1 occupancy in normal erythroid progenitors in both proliferating and differentiating conditions.
Project description:To study the gene expression changes during neuronal differentiation of mouse cerebellar granular progenitor cells (CGNPs), we generated neurospheres (proliferating CGNPs) and differentiated the cells (differentiating CGNPs). Then we performed RNA-Seq analysis.