Unknown,Transcriptomics,Genomics,Proteomics

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High Resolution Mapping of Mouse Embryonic Genome Activation using SMART-seq2 with in vitro polyadenylation

ABSTRACT: Single-embryo SMART-seq of mouse Embryonic Genome Activation with 2-cell stage embryos collected after 2 h precision in vitro fertilization (IVF) at the following timepoints: 17, 20, 23, and 26 h post-IVF. Individual embryos were lysed in total cell lysis buffer, and the lysates were split to perform in vitro polyadenylation on half of the sample. SMART-seq2 libraries were generated subsequently. All samples have matched in vitro polyadenylated (PolyA+) and nonadenylated (PolyA-) libraries.

INSTRUMENT(S): NextSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: Jasmina Al-Mousawi

PROVIDER: E-MTAB-16935 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:C/EBPβ plays a major role in numerous biological processes but unfortunately its precise role is not still clear and conflicting studies showed that this transcription factor could have contradictory functions. These latter arise from the complexity of mechanisms regulating C/EBPβ activity. Indeed, C/EBPβ encodes an intronless gene that generates a single mRNA that is alternatively translated into two major isoforms of 35kDa (liver-enriched activator protein: LAP) and 20kDa (liver-enriched inhibitory protein: LIP). LAP is the active isoform of this transcription factor whereas LIP, a truncated isoform negatively regulate C/EBPβ-LAP-mediated gene expression. The main goal of our research was to understand how LAP and LIP isoforms governe C/EBPβ cellular functions with the identification of new genes and pathways regulated by these isoforms in the human Hep3B hepatoma cell line. For this purpose an original in vitro system characterized by a target genes induction with the activatory C/EBPß isoform LAP and a target genes repression with the inhibitory C/EBPß isoform (LIP) was used to identify the genuine C/EBPβ molecular signature. Using a cDNA microarray which provides a complete coverage of the liver transcriptome, we identified 676 genes inversely regulated by LAP and LIP. These selected genes are involved in many biological processes as the hepatic metabolism (cholesterol), detoxification, induction of apoptosis and negative regulation of the cell proliferation. According to the involvement of C/EBPβ in hepatic carcinogenesis we focused on cell cycle regulation and apoptosis. Through functional studies, we proved for the first time that LIP plays in favor of the survival of the Hep3B cells whereas LAP makes the cells more sensitive to staurosporine-induced cell death. Moreover, a lot of studies demonstrated that the anti-proliferative action of C/EBPβ mainly depends on RB protein. By studying the rate of Hep3B cells proliferation which overexpress LAP, we brought to the fore that this isoform would be able to induce a repression of the Hep3B cells line proliferation - a RB- and p53- negative cell line. Thus, LAP seems able to induce the repression of proliferation by a different metabolic way from the RB one. Keywords: comparison of cells expressing LAP or LIP RNA were extracted from 5 Hep3BLAPexpressing LAP, 5 Hep3BLAP control without expression of LAP, 5 Hep3BLIP expressing LIP and 5 Hep3BLIP control without epression of LIP. Each sample was hybridized once in 5 different nylon membranes.

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High Resolution Mapping of Mouse Embryonic Genome Activation using SMART-seq2 with in vitro polyadenylation

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