Project description:Smad family proteins transduce signals downstream of transforming growth factor-beta (TGF-beta) and are one of the factors that regulate target genes related to diseases affecting the skin. We here identified C2orf54, officially known as MAB21L4, as one of the most up-regulated targets of TGF-beta and Smad3 in a differentiated human progenitor epidermal keratinocyte, using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). Smad2 and Smad3 bind to the regulatory regions of the C2orf54 gene locus. We found that TGF-beta induced expression of a barrier protein involucrin (encoded by IVL gene), and transcriptional activity of the IVL promoter induced by TGF-beta was inhibited by siRNAs for C2orf54. Further analysis revealed that C2orf54 siRNAs also down-regulated the expression of several target genes of TGF-beta. C2orf54 protein located mainly in the cytosol, physically bound to Smad2 and Smad3, but did not inhibit the binding of Smad2 and Smad3 to the target genomic regions. These findings suggested that TGF-beta-induced C2orf54 up-regulates gene expression induced by Smads, possibly through its physical interaction with Smad proteins.
Project description:Smad proteins transduce signals downstream of transforming growth factor-b (TGF-b), and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as one of the most up-regulated targets of TGF-b and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). Smad2 and Smad3 bound to the regulatory regions of the MAB21L4 gene locus. We found that TGF-b induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-b was inhibited by siRNAs for MAB21L4. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-b. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated acetylation of histone H3 lys 27 (H3K27ac), an active enhancer mark, near the Smad3 binding regions. These findings suggest that TGF-b-induced MAB21L4 up-regulates the gene expression induced by TGF-b, possibly through physical interactions with a transcriptional corepressor c-Ski in the cytosol.
Project description:Smad family proteins transduce signals downstream of transforming growth factor-beta (TGF-beta) and are one of the factors that regulate target genes related to diseases affecting the skin. We here identified C2orf54, officially known as MAB21L4, as one of the most up-regulated targets of TGF-beta and Smad3 in a differentiated human progenitor epidermal keratinocyte, using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). Smad2 and Smad3 bind to the regulatory regions of the C2orf54 gene locus. We found that TGF-beta induced expression of a barrier protein involucrin (encoded by IVL gene), and transcriptional activity of the IVL promoter induced by TGF-beta was inhibited by siRNAs for C2orf54. Further analysis revealed that C2orf54 siRNAs also down-regulated the expression of several target genes of TGF-beta. C2orf54 protein located mainly in the cytosol, physically bound to Smad2 and Smad3, but did not inhibit the binding of Smad2 and Smad3 to the target genomic regions. These findings suggested that TGF-beta-induced C2orf54 up-regulates gene expression induced by Smads, possibly through its physical interaction with Smad proteins.
Project description:Performing global gene expression profiling for different types of keratinocytes colonies can provide important information that might help in understanding the biology and ontogeny of epidermal skin stem cells. We used microarrays to detail the global gene expression profiling for different types of keratinocytes colonies including Holoclones (stem cells), Meroclones (transient amplifying cells), and Paraclones (differentiated cells) .
Project description:Human epidermal keratinocytes were treated with 25 ng.ml EphB2 or EFNA4, both as-Fc conjugates (Sigma). Human epidermal keratinocytes are treated with 25 ng/ml EphB2 or EFNA4 Fc conjugates in a 48hr time course.
Project description:Human induced pluripotent stem cells (iPSC) can be derived from patients or be genome-edited to introduce or correct disease-related genetic variants, and subsequently be differentiated into any skin cell type. Yet, the potential of induced keratinocytes (iKC) derived from iPSC has not been explored to model the chronic inflammatory skin disease atopic dermatitis (AD), partially because iKC are often immature and heterogeneous as compared to primary keratinocytes (pKC). We aimed to optimize iPSC to keratinocyte and subsequent epidermal differentiation protocols, and compared the response of iKC to inflammatory cytokines involved in the AD pathophysiology. We found that CELLnTEC (CnT)-30 medium greatly increased the iKC culture homogeneity based on morphology and transcriptome (e.g., 130-fold higher KRT5) as compared to commonly used keratinocyte serum free medium (KSFM). Single cell RNA sequencing of iKC revealed different cell populations in the iKC culture, and indicated cell surface markers to enrich for keratinocyte-like cells. In addition, iKC passaging enhanced overall keratinocyte marker gene expression like KRT14 to similar levels as primary keratinocytes (pKC) while maintaining the cobblestone keratinocyte morphology. Upon 2D calcium or fetal bovine serum (FBS) stimulation iKC showed flattened morphology and increased KRT1 and IVL expression, typical for epidermal differentiation. Epidermal differentiation markers were reduced again by AD cytokines IL-4, IL-13 and IL-22, mimicking the epidermis defects in AD. Finally, air exposure also induced 3D epidermal differentiation, including presence of all epidermal layers, and KRT10, IVL and FLG expression, while maintaining the proliferative capacity of keratinocytes in the basal layer based on Ki67 expression. Altogether, we show the potential of iPSC-derived keratinocytes to study epidermal defects in AD, which could facilitate patient-derived skin tissue modeling in translational research.
Project description:Targets of Retinoic Acid (RA) were identified in primary human epidermal keratinocytes grown in the presence or absence of all-trans retinoic acid for 1, 4, 24, 48 and 72 hours. Targets of Thyroid Hormone (T3) were identified in primary human epidermal keratinocytes grown in the presence or absence of the hormone; same controls as for RA.
Project description:Targets of Retinoic Acid (RA) were identified in primary human epidermal keratinocytes grown in the presence or absence of all-trans retinoic acid for 1, 4, 24, 48 and 72 hours. Targets of Thyroid Hormone (T3) were identified in primary human epidermal keratinocytes grown in the presence or absence of the hormone; same controls as for RA. Time course, 1, 4, 24, 48 and 72 hours