Investigating the influence of the homoebox transcription factor IRX1 on gene expression
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE75376 | GEO | 2016/11/14
SECONDARY ACCESSION(S): PRJNA304023
REPOSITORIES: GEO
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Project description:To explore the functon of IRX1 in gastric cancer, we employed whole genome microarray expression profiling as a discovery platform to identify gene expression changes in three samples. We constructed the eukaryotic expression vector pEGFP-IRX1. The pEGFP-IRX1 expression vector was transfected into SGC-7901 gastric cancer cells by Lipofectamine 2000. The IRX1 protein was mainly observed in nuclei. Naive SGC7901 and empty vector pEGFP-N1 transfected cells were used as controls.
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Project description:To explore the functon of IRX1 in gastric cancer, we employed whole genome microarray expression profiling as a discovery platform to identify gene expression changes in three samples. We constructed the eukaryotic expression vector pEGFP-IRX1. The pEGFP-IRX1 expression vector was transfected into SGC-7901 gastric cancer cells by Lipofectamine 2000. The IRX1 protein was mainly observed in nuclei. Naive SGC7901 and empty vector pEGFP-N1 transfected cells were used as controls. Gene expression profiles were compared between parental gastric cancer cell line SGC7901 and cells transfected with pEGFP-IRX1 and pEGFP-N1.
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Project description:The intrinsically photosensitive retinal ganglion cells (ipRGC) are the conduit between the retina and brain regions responsible for non-image-forming and image-forming vision. In mice, six ipRGC subtypes have been discovered based on morphological characteristics, functions, and molecular profiles. All ipRGCs arise from Tbr2-expressing RGCs during developmental stages and subsequently diverge and differentiate into the six mature, distinct subtypes in adult retinas. However, the cellular and molecular mechanisms controlling the formation and maturation of the six ipRGC subtypes remain elusive. Here, we demonstrate that two Tbr2-dependent transcription factors, Iroquois‑related homeobox 1 (Irx1) and T-box containing factor 20 (Tbx20), are key downstream transcription factors guiding lineage segregations of Tbr2-expressing RGC into distinct adult ipRGC subtypes. Both factors also control Opn4 expression. Irx1 is expressed in the M3, M4, and M5 subtypes, while Tbx20 is predominantly expressed in M1, M2, M6, and subgroups of M3 and M5. When Irx1 is ablated during retinal development, Opn4 expression is significantly reduced in the M3, M4, and M5 ipRGC groups; however, the formation of Irx1-expressing ipRGCs is not affected. In contrast, when Tbx20 is deleted, a significant number of Tbx20-expressing cells fail to develop while Opn4 expression is down-regulated. These findings reveal two parallel transcription cascades downstream of Tbr2 for controlling ipRGC subtype formation, fate divergence, and maintenance in the adult retina.
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Project description:Human induced pluripotent stem cells (hiPSCs) are useful as a tool for reproducing neural development in vitro. However, each hiPSC line has a different ability to differentiate into specific lineages, as known as differentiation propensity, resulting in reduced reproducibility and increased time and cost requirements for research use. To overcome this issue, we searched for predictive signatures of neural differentiation propensity of hiPSCs using DNA methylation which is the main modulator of cellular properties. We obtained 32 lines of hiPSC and its comprehensive DNA methylation data by Infinium MethylationEPIC beadchip. To assess the neural differentiation efficiency of these hiPSCs, we measured the percentage of PAX6-positive cells on day 7 of neural stem cell induction by the dual-SMAD inhibition protocol. Using DNA methylation data of undifferentiated hiPSCs and their measured differentiation efficiency into neural stem cells as the set of data, and HSIC Lasso, a machine learning-based nonlinear feature selection method, we attemted to identify neural differentiation associated differentially methylated sites. Epigenome-wide unsupervised clustering could not distinguish between hiPSCs with varying differentiation efficiency. On the other hand, HSIC Lasso identified 62 probes that can explain the neural differentiation efficiency of hiPSCs. Selected features by HSIC Lasso were particularly enriched within the 3 Mbp on chromosome 5, harboring the IRX2, C5orf38, and IRX1 genes. Within this region, DNA methylation rates were correlated with neural differentiation efficiency particular to female hiPSCs and negatively correlated with gene expression of the IRX1/2 genes. In addition, forced expression of the IRX1/2 genes impaired the neural differentiation ability of hiPSCs. We have shown for the first time that DNA methylation state on the IRX1/2 genes of hiPSCs is predictive biomarker of their ability for neural differentiation. The predictive markers for neural differentiation efficiency identified in this study can be useful for selection of suitable undifferetiated hiPSCs prior to differentiation induction.
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Project description:Human induced pluripotent stem cells (hiPSCs) are useful as a tool for reproducing neural development in vitro. However, each hiPSC line has a different ability to differentiate into specific lineages, as known as differentiation propensity, resulting in reduced reproducibility and increased time and cost requirements for research use. To overcome this issue, we searched for predictive signatures of neural differentiation propensity of hiPSCs using DNA methylation which is the main modulator of cellular properties. We obtained 32 lines of hiPSC and its comprehensive DNA methylation data by Infinium MethylationEPIC beadchip. To assess the neural differentiation efficiency of these hiPSCs, we measured the percentage of PAX6-positive cells on day 7 of neural stem cell induction by the dual-SMAD inhibition protocol. Using DNA methylation data of undifferentiated hiPSCs and their measured differentiation efficiency into neural stem cells as the set of data, and HSIC Lasso, a machine learning-based nonlinear feature selection method, we attemted to identify neural differentiation associated differentially methylated sites. Epigenome-wide unsupervised clustering could not distinguish between hiPSCs with varying differentiation efficiency. On the other hand, HSIC Lasso identified 62 probes that can explain the neural differentiation efficiency of hiPSCs. Selected features by HSIC Lasso were particularly enriched within the 3 Mbp on chromosome 5, harboring the IRX2, C5orf38, and IRX1 genes. Within this region, DNA methylation rates were correlated with neural differentiation efficiency particular to female hiPSCs and negatively correlated with gene expression of the IRX1/2 genes. In addition, forced expression of the IRX1/2 genes impaired the neural differentiation ability of hiPSCs. We have shown for the first time that DNA methylation state on the IRX1/2 genes of hiPSCs is predictive biomarker of their ability for neural differentiation. The predictive markers for neural differentiation efficiency identified in this study can be useful for selection of suitable undifferetiated hiPSCs prior to differentiation induction.
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Project description:Low transfection efficiency poses a significant challenge to experimental success, making the improvement of gene-carrying plasmid delivery and expression essential across various research fields. The stability of transfected vector DNA or mRNA significantly impacts transgene expression, as the innate immune system may recognize plasmid DNA as foreign and initiate a degradation response. Currently, it is unclear whether RNA transcribed from plasmids can activate RNA sensors, affecting transfection efficiency. This study employed RNA sequencing to analyze cellular responses to various circular and linear DNAs across five cell lines—HEK293T, HCT116, HeLa, L02, and NCM460—revealing that the innate immune response is a primary contributor to low transfection efficiency. Additionally, we used ChIP-seq to detect H3K27ac histone marks to explore whether the immune response triggered by plasmid transfection is regulated by epigenetic mechanisms. Finally, we performed ChIP assays for IRF7 and p-IRF3 in HeLa cells transfected with the linear pcDNA3.1-neo plasmid to demonstrate that these two transcription factors can indeed bind to the RTV genes.
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