Transcriptome analysis of iPSC-derived keratinocytes with or without a disease-associated TP63 mutation
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE109185 | GEO | 2018/01/16
SECONDARY ACCESSION(S): PRJNA430074
REPOSITORIES: GEO
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Project description:This transcriptome study is intended to discover the effects of MYC and TP63 on human keratinocytes differentiation at the genome level. Human keratinocyte cell line, HaCaT cells were transfected by siRNAs with the help of transfection reagent, INTERFERin. 48 hours post-transfection, total RNA was extracted from cells. We used a non-targeting siRNA as a negative control. siRNA targeting MYC or TP63 was obtained from QIAGEN, and RNAs from wild type HaCaT cells were used as reference samples. We did 3 biological replicates and 2 'dye swap' for each sample. Totally we got 12 samples, with 2 dye-swap for each sample, and 48 arrays used for all hybridization.
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Project description:Transcriptome analysis of iPSC-derived keratinocytes with or without a disease-associated TP63 mutation
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Project description:This transcriptome study is intended to discover the effects of MYC and TP63 on human keratinocytes differentiation at the genome level.
2009-07-30 | GSE17394 | GEO
Project description:The transcriptional basis for disrupted epidermal differentiation arising from TP63 AEC mutations remains to be elucidated. Here we present an organotypic model of AEC dysfunction that phenocopies differentiation defects observed in AEC patient skin. Transcriptional analysis of model AEC tissue revealed impaired induction of differentiation regulators, including OVOL1, GRHL3, KLF4, PRDM1 and ZNF750. Genome wide binding analyses of TP63 during epidermal differentiation showed direct binding of OVOL1, GRHL3, and ZNF750 promoters suggesting AEC mutants prevent normal activation of these targets by direct transcriptional interference. Remarkably, exogenous ZNF750 restores impaired epidermal differentiation caused by AEC mutation. Thus, repression of ZNF750 is central to disrupted epidermal differentiation in model AEC tissue. ChIP-Seq analysis: Examination of p63 binding in proliferating and differentiating human keratinocytes
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Project description:The transcriptional basis for disrupted epidermal differentiation arising from TP63 AEC mutations remains to be elucidated. Here we present an organotypic model of AEC dysfunction that phenocopies differentiation defects observed in AEC patient skin. Transcriptional analysis of model AEC tissue revealed impaired induction of differentiation regulators, including OVOL1, GRHL3, KLF4, PRDM1 and ZNF750. Genome wide binding analyses of TP63 during epidermal differentiation showed direct binding of OVOL1, GRHL3, and ZNF750 promoters suggesting AEC mutants prevent normal activation of these targets by direct transcriptional interference. Remarkably, exogenous ZNF750 restores impaired epidermal differentiation caused by AEC mutation. Thus, repression of ZNF750 is central to disrupted epidermal differentiation in model AEC tissue. Gene expression analysis: To establish a differentiation signature for primary human keratinocytes, with p63i-depleted, and ΔNp63α AEC mutants overexpressed, total RNA was isolated in biologic duplicate from cells in different conditions and hybridized to Affymetrix HG-U133 2.0 Plus arrays.
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Project description:The transcriptional basis for disrupted epidermal differentiation arising from TP63 AEC mutations remains to be elucidated. Here we present an organotypic model of AEC dysfunction that phenocopies differentiation defects observed in AEC patient skin. Transcriptional analysis of model AEC tissue revealed impaired induction of differentiation regulators, including OVOL1, GRHL3, KLF4, PRDM1 and ZNF750. Genome wide binding analyses of TP63 during epidermal differentiation showed direct binding of OVOL1, GRHL3, and ZNF750 promoters suggesting AEC mutants prevent normal activation of these targets by direct transcriptional interference. Remarkably, exogenous ZNF750 restores impaired epidermal differentiation caused by AEC mutation. Thus, repression of ZNF750 is central to disrupted epidermal differentiation in model AEC tissue.
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Project description:The transcriptional basis for disrupted epidermal differentiation arising from TP63 AEC mutations remains to be elucidated. Here we present an organotypic model of AEC dysfunction that phenocopies differentiation defects observed in AEC patient skin. Transcriptional analysis of model AEC tissue revealed impaired induction of differentiation regulators, including OVOL1, GRHL3, KLF4, PRDM1 and ZNF750. Genome wide binding analyses of TP63 during epidermal differentiation showed direct binding of OVOL1, GRHL3, and ZNF750 promoters suggesting AEC mutants prevent normal activation of these targets by direct transcriptional interference. Remarkably, exogenous ZNF750 restores impaired epidermal differentiation caused by AEC mutation. Thus, repression of ZNF750 is central to disrupted epidermal differentiation in model AEC tissue.
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Project description:Our goal was to demonstrate the similarity between the original keratinocytes and iPSC-derived keratinocytes from the same individual Profiles of original keratinocytes, iPSCs, IPSC-derived keratinocytes, iPSC-derived fibroblasts from a single individual and normal fibroblasts from a second individual were compared
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Project description:Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional Type VII collagen and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma (SCC). The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We have formed a multidisciplinary team with the ultimate goal to develop an iPSC-based therapy for RDEB. Here, we present a clinical protocol that generates autologous, corrected epithelial keratinocyte sheets with the COL7A1 gene mutation corrected for grafting on to patients. We demonstrate the utility of sequential reprogramming and novel adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity and secreted wild-type collagen VII, resulting in stratified epidermis in vitro and in vivo in mice. Sequencing of corrected cell lines prior to tissue formation revealed heterogeneity of SCC-predisposing mutations, allowing us to select COL7A1 corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a first clinical platform to use iPSCs in the treatment of debilitating genodermatoses. Microarray analysis of iPS-derived keratinocytes from two RDEB patients (iPS-K1 and iPS-K3), corresponding patient keratinocytes (AHK1 and AHK2), normal human keratinocytes (NHK), as well as H9 human embryonic stem cells (hESC - H9). All samples were analyzed in duplicate and differential gene expression was measured relative to H9.
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