Unknown,Transcriptomics,Genomics,Proteomics

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Temporal Endogenous Gene Expression Profiles in Response to Lipid-Mediated Transfection

ABSTRACT: The current understanding of the molecular factors underlying LF2000-mediated transfection is largely unknown. Cationic LF2000 gene delivery system was used to transfer GFP transgene to HEK293T cells. FACS separation of transfected (GFP positive), untransfected (GFP negative), and untreated cells enabled gene expression profiles to be obtained using Affymetrix® HG-U133A 2.0 microarrays for each cell population. Gene profiles were differentially compared for each population combination. Control (untreated), transfected (GFP positive), and untransfected (GFP negative) RNA samples were purified from HEK293T cells after FACS isolation. Treated experiments were repeated on triplicate days to run 3 microarrays for each cell population. Controls were run on duplicate to give 2 microarrays for each population.

ORGANISM(S): Homo sapiens

SUBMITTER: Timothy Martin

PROVIDER: E-GEOD-59666 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Neural basic helix-loop-helix (bHLH) transcription factors are important for the differentiation and cell type specification of neurons. They are thought to share direct downstream targets in their common role as neuronal differentiation factors, but have distinct targets with respect to their cell type specific roles. Little is known about distinct cell-type specific bHLH targets as previous work did not distinguish these from common targets. Based on previous genetic evidence, we hypothesize that bHLH transcription factors have unique targets for their function in regulating neuronal sub-type specification. Atoh1 (Math1) is a bHLH transcription factor that specifies different cell types of the proprioceptive pathway in mammals such as the dorsal interneuron 1 population of the developing neural tube. Using microarray analyses of neighboring specific bHLH sorted populations from developing mouse neural tubes, we determine transcripts unique to the Atoh1-derived population and not those common to bHLH transcription factors in related neural progenitor populations. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments of native tissue followed by enhancer reporter analyses identified five direct cell-type specific targets of Atoh1 in vivo: Klf7, Rab15, Rassf4, Selm, and Smad7, along with their Atoh1-responsive enhancers. These Atoh1 targets were found from native tissue in the appropriate developmental context and have diverse functions that range from transcription factors to regulators of endocytosis and signaling pathways. Only Rab15 and Selm are expressed across several different Atoh1-specified cell types including external granule cells (EGL) in the developing cerebellum, hair cells of the inner ear, and Merkel cells, demonstrating that even within Atoh1 lineages, not all Atoh1 specific targets are shared. Our work establishes on a molecular level that the neuronal differentiation bHLH transcription factors also have distinct targets for their roles in neuronal sub-type specification. From this work, we can begin to address how bHLH transcription factors are able to specify unique cell types and initiate programs that organize neuronal diversity. Gene expression analysis: Two samples, Atoh1-GFP and dNeurog1-GFP, were analyzed. Two biological replicates of each. Atoh1-GFP transgenics are mice with GFP inserted into a BAC that drives GFP to the Atoh1-derived population of the developing neural tube marking the dorsal interneuron 1 population (Raft et al. Development 2007). dNeurog1-GFP transgenics are mice with a Neurog1 enhancer that drives GFP to the dorsal part of the developing neural tube marking the dorsal interneuron 2 population (Nakada et al. Dev Bio 2004). Chip-seq analysis: Series GSE22111

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Temporal Endogenous Gene Expression Profiles in Response to Lipid-Mediated Transfection

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