ABSTRACT: HNF4α and TCF4 (TCF7L2) ChIPseq in Tet-On HCT116 inducible cell lines that express either the human HNF4α2 or HNF4α8 under the control of doxycycline (DOX) [ChIP-Seq]
Project description:Purpose: Aim of the study is to determine how many of the dysregulated genes in the RNAseq are direct targets of (P1) HNF4α2 and (P2) HNF4α8 and examine HNF4α and TCF4 binding in vivo. We performed ChIPseq on TCF4 in the absense or presence of DOX in the Tet-On inducible HCT116 HNF4α2 and HNF4α8 lines, and ChIPseq for HNF4α (a445 Ab) in the presence of DOX. Methods: HNF4α2 and HNF4α8 lines were induced with 0.3 μg/mL DOX for 24 hours. Samples were generated by deep sequencing, using the NEXTflex ChIPseq. Result: There were more HNF4α2 peaks than HNF4α8 peaks, with some common peaks bound by HNF4α2 and HNF4α8. Binding patterns were observed between HNF4α and TCF4. Conclusion: HNF4α2 can displace TCF4 better than HNF4α8 on AP-1 bound sites. Tet-On inducible HCT116 cell (HNF4α2 and HNF4α8) lines, treated with (0.3 μg/mL) or without DOX for 24 hours, were 50bp single-end sequenced using Illumina-compatible-NEXTflex ChIP kit (Bioo Scientific).
Project description:Purpose: Aim of the study is to determine how many of the dysregulated genes in the RNAseq are direct targets of (P1) HNF4α2 and (P2) HNF4α8 and examine HNF4α and TCF4 binding in vivo. We performed ChIPseq on TCF4 in the absense or presence of DOX in the Tet-On inducible HCT116 HNF4α2 and HNF4α8 lines, and ChIPseq for HNF4α (a445 Ab) in the presence of DOX. Methods: HNF4α2 and HNF4α8 lines were induced with 0.3 μg/mL DOX for 24 hours. Samples were generated by deep sequencing, using the NEXTflex ChIPseq. Result: There were more HNF4α2 peaks than HNF4α8 peaks, with some common peaks bound by HNF4α2 and HNF4α8. Binding patterns were observed between HNF4α and TCF4. Conclusion: HNF4α2 can displace TCF4 better than HNF4α8 on AP-1 bound sites.
Project description:Purpose: Aim of the study is to identify functional differences between the P1 and P2-HNF4α isoforms. To do this, we generated Tet-On inducible lines that express either the human (P1) HNF4α2 or (P2) HNF4α8 under control of DOX in the HCT116 human colon cancer cells. Methods: HNF4α2 and Parental lines were induced with 0.3 μg/mL DOX, while HNF4α8 line was induced with either 0.1 or 0.3 μg/mL DOX for 24 hours. Samples were generated by deep sequencing, using the Illumina TruSeq RNA. Result: There were common and unique dysregulated genes identified in the HNF4α2 and HNF4α8 lines (+DOX); more upregulated genes than downregulated genes in both the lines. Conclusion: The functional difference between HNF4α2 and HNF4α8 is that the latter tends to upregulate genes involved in proliferation and anti-apoptosis while HNF4α2 upregulates genes involved in growth suppression and cell death. Tet-On inducible HCT116 cell (Parental, HNF4α2, and HNF4α8) lines, treated with (0.0, 0.1, or 0.3 μg/mL) DOX for 24 hours, were 50bp pair-ended sequenced in triplicate using Illumina TruSeq RNA Sample Prep v2 Kit.
Project description:Purpose: Aim of the study is to identify functional differences between the P1 and P2-HNF4α isoforms. To do this, we generated Tet-On inducible lines that express either the human (P1) HNF4α2 or (P2) HNF4α8 under control of DOX in the HCT116 human colon cancer cells. Methods: HNF4α2 and Parental lines were induced with 0.3 μg/mL DOX, while HNF4α8 line was induced with either 0.1 or 0.3 μg/mL DOX for 24 hours. Samples were generated by deep sequencing, using the Illumina TruSeq RNA. Result: There were common and unique dysregulated genes identified in the HNF4α2 and HNF4α8 lines (+DOX); more upregulated genes than downregulated genes in both the lines. Conclusion: The functional difference between HNF4α2 and HNF4α8 is that the latter tends to upregulate genes involved in proliferation and anti-apoptosis while HNF4α2 upregulates genes involved in growth suppression and cell death.
Project description:AGY571 cells engineered to express Dox-inducible GFP or ATG7 were treated with 500 ng/mL or 1 ug/mL Doxycycline for 48 hours. Assessing the effects of ATG7 reexpression in B cell lymphoma.
Project description:β-catenin signaling can be both a physiological and an oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20 to 40% of hepatocellular carcinomas with specific metabolic features. We decipher the molecular determinants of β-catenin-dependent zonal transcription using mice with β-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by Tcf4 and β-catenin, their transcriptome and their metabolome. We find that Tcf4 DNA-bindings depend on β-catenin. Tcf4/β-catenin binds Wnt-responsive elements preferentially around β-catenin-induced genes. In contrast, genes repressed by β-catenin bind Tcf4 on Hnf4-responsive elements. β-catenin, Tcf4 and Hnf4α interact, dictating β-catenin transcription which is antagonistic to that elicited by Hnf4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by β-catenin, partly through xenobiotic nuclear receptors. We conclude that β-catenin patterns the zonal liver together with Tcf4, Hnf4α and xenobiotic nuclear receptors. This network represses lipid metabolism, and exacerbates glutamine, drug and bile metabolism, mirroring hepatocellular carcinomas with β-catenin mutational activation. In vivo liver samples in 4 conditions: Betacat activated (WCE, Tcf4 chipseq, Betacat chipseq, mRNAseq with 2 replicates), Betacat null (WCE, Tcf4 chipseq, mRNAseq with 2 replicates), Betacat control (mRNAseq with 2 replicates), Wild type (mRNAseq with 2 replicates)
Project description:We performed microarray analysis on BT549 cells that express miR-200c in a doxycycline (dox)-inducible manner from the miR-200c-TripZ system.
Project description:We performed RNA-seqeuncing analysis on mouse Met-1 cells that express miR-200c in a doxycycline (DOX)-inducible manner from the miR-200c-TripZ system.
Project description:Gene expression of T47D-MTVL human breast cancer cells expressing Dox-inducible shRNAs against histone H1.4 (sh120) or multiple H1 variants (sh225), or overexpressing WT or K26A mutant HA-tagged H1.4. T47D-MTVL, breast cancer cell line carrying one stably integrated copy of luciferase reporter gene driven by the MMTV promoter, is stably infected with an inducible system for the expression of shRNAs.Cells stably express RedFP and KRAB repressor fused to Tet regulator.Upon Dox treatment, cells express RedFP and the cloned shRNA. Stable breast cancer-derived cell lines expressing an shRNA against one of each of the histone H1 isoforms in response to doxycycline (Dox) were grown for six days in the presence or absence of Dox, in duplicate, and RNA extracted for microarray hybridization. Cell lines used: inducible shRNA against H1.4 or multiple H1 variants, and random shRNA-expression vector. Stable breast cancer-derived cell lines expressing the histone H1.4 isoform, WT or K26A, HA-tagged, in duplicate, and RNA extracted for microarray hybridization. Cell lines used: overexpressing H1.4 WT or K26A mutant.
Project description:To reprogram adult HSPCs in vivo, we used a mouse engineered to express LIN28B in a Doxycycline (Dox)-inducible manner (iLIN28B). We performed single cell RNA-seq to compare the transcriptomes of common lymphoid progenitor (CLP) cells isolated from mouse FL, adult BM of wildtype mice, adult BM of iLIN28B treated with Dox (+Dox), and adult BM of littermate controls +Dox.
