KLF7 ChIP-seq in human keratinocytes HaCaT cell line
Ontology highlight
ABSTRACT: KLF7, a member of the KLF family, is an evolutionarily conserved zinc finger-containing transcription factor. Previous studies demonstrated that KLF7 possesses diverse regulatory functions related to embryogenesis, cell growth, proliferation, and differentiation. Our results reveal that there was an increased abundance of KLF7 in OSM-treated HaCaT cells. Mechanistically, our results showed that OSM induces epidermal keratinocyte differentiation through phosphorylation of STAT5, which binds to the promoter and activates KLF7 transcription.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling (RNA-seq) between HaCaT cell and OSM-treated HaCaT cells Methods: mRNA profiles of HaCaT cells and OSM-treated HaCaT cells were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks.
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
Project description:The p53 protein is encoded by TP53 gene and plays the key role in significant number of cellular processes including proliferation, apoptosis and regulation of many stress response pathways. P53 acts like a direct transcription activator of numerous genes regulating cell cycle arrest, DNA repair, growth inhibition and many others (Mollereau and Ma, 2014). The canonical biological function of p53 is maintaining genome integrity via elimination of damaged or exposed to genotoxic stress cells. Immortalized HaCaT cells are widely used for keratinocyte research, since they maintain stable keratinocyte phenotype, have nearly unlimited proliferative potential, do not require specific growth and differentiation factors (Colombo et al., 2017). Also, HaCaT cells produce typical differentiation markers such as cytokeratins K14 and K10, involucrin (Colombo et al., 2017) and respond to keratinocyte differentiation stimuli. Taking together, HaCaT cells have similar to normal human keratinocytes (NHK) properties, however, as many of spontaneously immortalized cell lines HaCaT cells bear two mutant p53 alleles - R282Q and H179Y (Lehman et al., 1993). Mutp53 in HaCaT has an increased affinity to other p53 family members (p63, p73), which significantly expands p53 properties. Moreover, mutp53 indirectly affects specific target genes via protein-protein interactions with other transcription factors (NF-Y, E2F1, NF-KB) or by tethering p63 to new promotor locations. For more detailed investigation of mutp53 impact on various processes in HaCaT cells we performed a shRNA mediated knockdown of mutp53. For generation of stable TP53 knockdown we employed plasmid vector pLKO-p53-shRNA-941 (Addgene # #25637) followed by puromycin selection of transduced cells. Here we present proteomic dataset obtained from wild type HaCaT cells and p53 knock down HaCaT keratinocytes.
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 Keywords: repeat sample
Project description:While the regulatory landscape during stem cell differentiation has been well characterized, the shared and unique regulatory mechanisms in different ectodermally-derived epithelial cells have not been well described. Through defining the complement of super enhancers and typical enhancers in corneal epithelium for the first time, we show that regulatory regions are often shared between cell types of the same lineage, and that corneal super enhancers are already marked as potential regulatory domains in embryonic stem cells. Through the enrichment of KLF motifs in enhancers, we identified and defined a novel role for Kruppel family member KLF7 in promoting the corneal progenitor cell state, in many cases working antagonistically to corneal differentiation promoting KLF4. Our work highlights the importance of balance between proliferation and differentiation, both for proper tissue development and for homeostasis.
Project description:While the regulatory landscape during stem cell differentiation has been well characterized, the shared and unique regulatory mechanisms in different ectodermally-derived epithelial cells have not been well described. Through defining the complement of super enhancers and typical enhancers in corneal epithelium for the first time, we show that regulatory regions are often shared between cell types of the same lineage, and that corneal super enhancers are already marked as potential regulatory domains in embryonic stem cells. Through the enrichment of KLF motifs in enhancers, we identified and defined a novel role for Kruppel family member KLF7 in promoting the corneal progenitor cell state, in many cases working antagonistically to corneal differentiation promoting KLF4. Our work highlights the importance of balance between proliferation and differentiation, both for proper tissue development and for homeostasis.
Project description:Increased expression of Kruppel like factor 7 (KLF7) is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia. The contribution of KLF7 to hematopoiesis has not been previously described. Herein, we characterized the effect on murine hematopoiesis of the loss of KLF7 and enforced expression of KLF7. Long-term multilineage engraftment of Klf7-/- cells was comparable to control cells, and self-renewal, as assessed by serial transplantation, was not affected. Enforced expression of KLF7 results in a marked suppression of myeloid progenitor cell growth and a loss of short- and long-term repopulating activity. Interestingly, enforced expression of KLF7, while resulting in multi-lineage growth suppression that extended to hematopoietic stem cells and common lymphoid progenitors, spared T cells and enhanced the survival of early thymocytes. RNA expression profiling of KLF7-overexpressing hematopoietic progenitors identified several potential target genes mediating these effects. Notably, the known KLF7 target Cdkn1a (p21Cip1/Waf1) was not induced by KLF7, and loss of CDKN1A does not rescue the repopulating defect. These results suggest that KLF7 is not required for normal hematopoietic stem and progenitor (HSPC) function, but increased expression, as seen in a subset of lymphoid leukemia, inhibits myeloid cell proliferation and promotes early thymocyte survival. KLF7 overexpression in HSPCs expression array: Lin- c-Kit+ Sca-1+ cells transduced with a KLF7 expressing or control (empty vector) lentivirus. Expression profiles of KLF7 overexpressing vs controls HSPCs. Cells were harvested 72 hrs post-transduction to compare expression profiles of control vs KLF7 overexpressing HSPCs
Project description:While the regulatory landscape during stem cell differentiation has been well characterized, the shared and unique regulatory mechanisms in different ectodermally-derived epithelial cells have not been well described. Through defining the complement of super enhancers and typical enhancers in corneal epithelium for the first time, we show that regulatory regions are often shared between cell types of the same lineage, and that corneal super enhancers are already marked as potential regulatory domains in embryonic stem cells. Through the enrichment of KLF motifs in enhancers, we identified and defined a novel role for Kruppel family member KLF7 in promoting the corneal progenitor cell state, in many cases working antagonistically to corneal differentiation promoting KLF4. Our work highlights the importance of balance between proliferation and differentiation, both for proper tissue development and for homeostasis.