Project description:Ehf is a transcriptional regulator that is highly expressed and enriched in corneal epithelium. To gain insights into the role of Ehf in the corneal epithelium, we performed siRNA knockdown of Ehf in primary human corneal epithelial cells. Primary human corneal epithelial cells were transfected with siEhf or si controls, plated, and harvested at 72 hr.
Project description:Ehf is a transcriptional regulator that is highly expressed and enriched in corneal epithelium. To gain insights into the role of Ehf in the corneal epithelium, we performed siRNA knockdown of Ehf in primary human corneal epithelial cells.
Project description:The cornea, composed of epithelium, stroma and endothelium, protects the anterior compartment of the eye from damage and allows transmission of light into the eye. While well described morphologically, no studies have investigated the global gene expression changes in the cornea throughout the mouseM-bM-^@M-^Ys life. We characterized the global gene expression profile of mouse cornea from early development through aging, and compared to gene expression in other epithelial tissue, to identify cornea enriched genes, pathways, and transcriptional regulators. We identified Ehf, an ets family transcription factor, as being highly selectively expressed in the corneal epithelium compared to the stroma, and highly expressed in cornea compared to other epithelial tissues. siRNA experiments and Ehf ChIP-Seq on mouse corneal epithelium confirm the role of this factor in promoting epithelial identity and cell differentiation, and suggest it carries out these functions through interactions with other cornea epithelial differentiation factors including Klf4. Whole eye globes were dissected from wild type CB6 mice. Corneal epithelium was isolated by digestion in 50% EMEM/dispase II with 50 mM sorbitol for two hours at 37M-BM-0C. ChIP was performed with an Ehf antibody, and was sequenced with an input control.
Project description:Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knockdown also targeted genes in pathways of epithelial development and differentiation and locomotory behavior. mRNA profiles from Calu-3 cells treated with negative control (NC) or EHF siRNA, in quintuplicate. mRNA profiles from 3 pcDNA (empty vector control) clones and 3 pcDNA-EHF overexpression A549 clones, 3-4 replicates each.
Project description:The cornea, composed of epithelium, stroma and endothelium, protects the anterior compartment of the eye from damage and allows transmission of light into the eye. While well described morphologically, no studies have investigated the global gene expression changes in the cornea throughout the mouse’s life. We characterized the global gene expression profile of mouse cornea from early development through aging, and compared to gene expression in other epithelial tissue, to identify cornea enriched genes, pathways, and transcriptional regulators. We identified Ehf, an ets family transcription factor, as being highly selectively expressed in the corneal epithelium compared to the stroma, and highly expressed in cornea compared to other epithelial tissues. siRNA experiments and Ehf ChIP-Seq on mouse corneal epithelium confirm the role of this factor in promoting epithelial identity and cell differentiation, and suggest it carries out these functions through interactions with other cornea epithelial differentiation factors including Klf4.
Project description:To understand the gene-regulatory role of H19 in corneal epithelial cells and to explore whether CLIM activation of H19 might account for non-cell proliferation effects of CLIM, we used siRNA to knock down H19 in primary human corneal epithelial cells. Microarray gene expression analysis revealed that 1,249 genes are down regulated 1.5 fold or more by siH19 compared to scramble control, and that 944 genes are up regulated
Project description:Genome-wide analysis of dihydrotestosterone (DHT) induced changes in gene expression in primary and immortalized human corneal epithelial cells. Analysis of regulation of primary and immortalized human corneal epithelial cells by dihydrotestosterone at gene expression level. The hypothesis tested in the present study was that the androgen-eye interaction in ocular surface epithelial cells like corneal cells is influenced by androgens through regulation of the expression of multiple genes. Results provide important information of the differential regulation and comparitive analysis of numerous genes in response to dihydrotestosterone incubation in primary and immortalized human corneal epithelial cells. Total RNA was obtained from primary and immortalized human corneal epithelial cells treated for 5 days with 10 nM dihydrotestosterone (n=3) or vehicle (n=3). The RNA was then used with Illumina HumanHT-12 v3 Expression BeadChips to determine the effect of DHT on gene expression in the primary human corneal cells grown in our laboratory and the immortalized human corneal epithelial cell line developed in Dr. Rheinwald's laboratory [Rheinwald et al. MCB, 22 (14): 5157. (2002)] and charecterized in Dr. Ilene Gibson's laboratory [Gipson et al. IOVS, 44 (6): 2496. (2003)].
Project description:PURPOSE. Myeloma Overexpressed gene (MYEOV) was initially identified as a gene amplified in several malignancies, and it was found to promote cell proliferation and metastasis. Our previous comparative RNA-seq and epigenetic analyses revealed high MYEOV levels in differentiated corneal epithelial cells and showed that MYEOV expression was epigenetically regulated by TET2. In the current study, we aimed to characterize further the expression and regulation of MYEOV in the human ocular surface epithelium. METHODS. MYEOV expression was examined by immunostaining and publicly available single-cell RNA-seq data. Gene knockdown (KD) of MYEOV and the regulators of corneal epithelial differentiation, PAX6 and KLF4, in in vitro-expanded corneal epithelial cells was performed by siRNA transfection. Protein expression levels were examined by Western blot. MYEOV KD cells were subjected to colony-forming assay and RNA-seq analysis. RESULTS. Human cornea immunostaining revealed high MYEOV expression in the nuclei of KRT12-positive differentiated corneal epithelial cells, while KRT13-positive differentiated conjunctival epithelial cells were MYEOV-negative. MYEOV expression was not detected in the other surface ectoderm-derived epithelia: epidermis and oral mucosa. Both PAX6 KD and KLF4 KD led to the reduction of MYEOV and KRT12 protein expression. MYEOV KD decreased colony-forming efficiency while afflicting limited global gene expression change. CONCLUSIONS. Our study revealed specific MYEOV expression in KRT12-positive corneal epithelial cells among surface ectoderm-derived epithelia. Similar to KRT12, MYEOV expression is regulated by PAX6 and KLF4. Functionally, MYEOV regulates cell proliferation of corneal epithelial cells.
Project description:Genome-wide analysis of dihydrotestosterone (DHT) induced changes in gene expression in primary and immortalized human corneal epithelial cells. Analysis of regulation of primary and immortalized human corneal epithelial cells by dihydrotestosterone at gene expression level. The hypothesis tested in the present study was that the androgen-eye interaction in ocular surface epithelial cells like corneal cells is influenced by androgens through regulation of the expression of multiple genes. Results provide important information of the differential regulation and comparitive analysis of numerous genes in response to dihydrotestosterone incubation in primary and immortalized human corneal epithelial cells.