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:The specific functional features of the epidermal keratinocytes are determined by the activity of many genes. The aim of the project was to characterize the role of HSPA2, a member of the HSPA chaperone family (HSP70), in human epidermal keratinocytes. The inactivation of the HSPA2 gene in the HaCaT line of spontaneously immortalized epidermal keratinocytes was performed by CRISPR/Cas9 gene editing system. Next, the effect of modifications on the transcriptomic profile of cells growing in a three-dimensional model of reconstructed human epidermis in vitro was investigated. The cells were grown at air-liquid interface culture on collagen-fibroblast matrix to achieve maximal level of HaCaT differentiation in RHE system.
Project description:The specific functional features of the epidermal keratinocytes are determined by the activity of many genes. The aim of the project was to characterize the role of HSPA2, a member of the HSPA chaperone family (HSP70), in human epidermal keratinocytes. The inactivation of the HSPA2 gene in the HaCaT line of spontaneously immortalized epidermal keratinocytes was performed by CRISPR/Cas9 gene editing system. Next, the effect of modifications on the transcriptomic profile of cells growing in 2D monolayer culture was investigated. This study was supported by a Polish National Science Center grant number NCN 2017/25/B/NZ4/01550.
Project description:The specific functional features of the epidermal keratinocytes are determined by the activity of many genes. The aim of the project was to characterize the role of HSPA2, a member of the HSPA chaperone family (HSP70), in human epidermal keratinocytes. The downregulation of HSPA2 gene in HaCaT cell line was performed by lentivirus-mediated shRNA method. Next, the effect of modifications on the transcriptomic profile of cells growing in a three-dimensional model of reconstructed human epidermis in vitro was investigated. The cells were grown at air-liquid interface culture on extracellular matrix to achieve maximal level of HaCaT differentiation in RHE system. This study was supported by a Polish National Science Center grant number NCN 2017/25/B/NZ4/01550.
Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5µg/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide
Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5µg/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide
Project description:We employed human HaCaT cells as a model system to identify cellular proteins that accompany SDS-induced toxicity based on a proteomic approach. HaCaT human keratinocyte cell line were treated with a non-cytotoxic dose of SDS (25 µg/ml, as determined by the MTT assay and microscopically examination) for 48 h. The altered abundance of proteins from HaCaT keratinocytes exposed to SDS was analyzed by LC-MS/MS approach and quantified using Progenesis LC software. The abundance of 217 proteins (which were identified by multiple peptides, ≥ 2) was altered in keratinocytes exposed to SDS; in which 131 proteins had increased abundance while 86 proteins was down regulated. The Pathview map of 131 up-regulated proteins was built and enhancement of glycolysis/gluconeogenesis was found.
Project description:The keratinocyte cell line HaCaT was cultured for three days (proliferation) or for ten days (differentiation). RNA from cells at day3 was compared to RNA from cells at day10. The microarray hybridizations were performed in dye-swap procedure : RNA from cells at day3 was labeled with Cy3 (GSM4674, GSM4675, GSM4682, GSM4683) and then with cy5 (GSM4680, GSM4681). Keywords = cell density differentiation program in human keratinocytes