HNF4α transduction and 5'azacytidine treatment of rat liver progenitor cells (rLEC) to promote the hepatic phenotype
ABSTRACT: Stem/progenitor cells represent an unlimited cell source for the generation of hepatocytes. As such, it was already shown that the hepatic liver progenitor-like cell line rLEC acquires a hepatocyte-like phenotype upon sequential exposure to hepatic growth factors and cytokines. However, expression of hepatocyte nuclear factor (HNF) 4α is still missing, hampering their full differentiation. Therefore, we aim at studying the phenotype of rLEC by transducing the cells with HNF4α. In addition, to make the genes more sensitive to transcription and differentiation, the effect of the DNA methyl transferase inhibitor 5' azacytidine (AZA) (20 µM) was also investigated. The global expression profiles of both microRNA and mRNA were studied by means of microarray technology. Overall design: rLEC were transduced with a viral vector containing HNF4α and puromycin selected. In addition, cells were exposed to 20µM 5'azacytidine for 7 days
INSTRUMENT(S): [miRNA-4] Affymetrix Multispecies miRNA-4 Array [ProbeSet ID version]
Project description:The histone deacetylase HDAC3 is a critical mediator of hepatic lipid metabolism, and liver-specific deletion of HDAC3 leads to fatty liver. To elucidate the underlying mechanism we developed a method of cross-linking followed by mass spectrometry to define a high-confidence HDAC3 interactome in vivo that includes the canonical NCoR/HDAC3 complex as well as Prospero-related homeobox 1 protein (PROX1). HDAC3 and PROX1 co-localize extensively on the mouse liver genome, and are co-recruited by Hepatocyte Nuclear Factor 4α (HNF4α). The HDAC3-PROX1 module controls the expression of a gene program regulating lipid homeostasis, and hepatic-specific ablation of either component increases triglyceride content in liver. These findings underscore the importance of specific combinations of transcription factors and coregulators in the fine tuning of organismal metabolism. Overall design: Biological replicates were uploaded in separated files and indicated in the file names.
Project description:Hepatocyte nuclear factor 4alpha (HNF4α) is a nuclear receptor with an emerging role in the gut. While HNF4α has been implicated in colitis and colon cancer in humans, deciphering its functional role is complicated by the existence of two promoters (P1 and P2) in theHNF4A gene that drive the expression of multiple isoforms in the adult intestine. In this study we investigate the roles of P1- and P2-driven HNF4α under conditions of homeostasis, colitis and colitis-associated colon cancer (CAC). P1- and P2-HNF4α are differentially expressed in the differentiated and proliferative compartments of the normal colonic crypt, respectively. Expression profiling of untreated exon swap mice suggests distinct functions of the isoforms that were corroborated in migration and ion transport assays. Overall design: To investigate the function of the HNF4α isoforms in the colon we utilized HNF4α isoform-specific mice developed previously by an exon swap strategy (PMID: 16498401). WT mice express P1-HNF4α predominantly at the top of the crypt in the differentiated compartment and P2-HNF4α in the bottom, proliferative compartment. The exon swap mice express only P1-HNF4α (α1 HMZ) or P2-HNF4α (α7 HMZ) throughout the colonic crypt. To identify functional differences between the isoforms we performed microarray analysis on RNA isolated from the distal colon of untreated young adult males and found a significant change in a substantial number of genes in the isoform-specific mice compared to their respective WT controls
Project description:A previous study from this laboratory demonstrated that up-regulating HNF4a could reverse the malignant phenotypes of HCC by inducing redifferentiation of HCC cells to hepatocytes. To study the mechanisms of the hepatic differentiation effect by HNF4α, we used the cDNA microarray to detect differential gene expression profiles of Hep3B infected with AdHNF4α and AdGFP. Expression profile analysis revealed that HNF4α positively regulated 1218 mRNAs and negatively regulated 1411 mRNAs for more than 2 times. The pathway analysis for the differential genes showed that the genes were involved in Complement and coagulation cascades, metabolism, Type II diabetes mellitus, Pathways in cancer etc. Overall design: Hepatoma cell lines Hep3B cells were seeded onto culture dishes and infected with AdHNF4α and AdGFP (as a control) at MOI 100. After Hep3B was infected by AdHNF4α or AdGFP for 72 hours, the cells were collected for cDNA Microarray analysis.
Project description:Background & Aims: The role of HNF4α has been extensively studied in hepatocytes and pancreatic β cells, but emerging evidence indicates that HNF4α is a key regulator of intestinal epithelial cell differentiation as well. The aim of the present work is to identify HNF4α target genes in the intestine in order to elucidate the role of HNF4α in differentiation of the intestinal epithelial cells. Results: One thousand one hundred and seventy-six genes were identified as HNF4α targets, many of which have not previously been described as being regulated by HNF4α. The 1,176 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. A thorough analysis of Cdx-2, trehalase, and cingulin promoters verified that these genes are regulated by HNF4α. In each case we were able to identify a functional HNF4α binding site in their promoters. Conclusions: HNF4α regulation of the Cdx-2 promoter unravels a transcription factor network also including HNF1α and β, all of which are transcription factors involved in intestinal development and gene expression. Keywords: ChIP-CHIP and expression data Overall design: Methods: We have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4α binding to promoter regions. The HNF4α ChIP-chip data was matched with gene expression and histone H3 acetylation status of the promoters in order to identify HNF4α binding to actively transcribed genes with an open chromatin structure.
Project description:5-Azacytidine or 5-aza-2’-deoxycytidine is a chemical analogue of the cytosine nucleoside used in DNA and RNA. Cells in the presence of 5-azacytidine incorporate it into DNA during replication and RNA during transcription. The incorporation of 5-azacytidine into DNA or RNA inhibits methyltransferase thereby causing demethylation in that sequence, affecting the way that cell regulation proteins are able to bind to the DNA/RNA substrate. Inhibition of DNA occurs through the formation of stable complexes between the molecule and with DNA methyltransferases, thereby saturating the cells methylation machinery. 5-azacytidine is mainly used in the treatment of myelodysplastic syndrome. Changes in gene expression levels in Erbb2 transfected murine fibroblast cell line after treatment with Azacytidine in two different concentrations were analysed. Keywords: cDNA microarray, murine fibroblast, azacytidine treatment, carcinogenesis Overall design: Gene expression levels of Erbb2 transfected NIH3T3 cell lines treated with 5µM and 10µM Azacytidine were compared to Erbb2 transfected but non-treated NIH3T3 cell line. For each Azacytidine concentration 4-8 experiments were performed including 50% dye swaps. As a controll experiments gene expression levels of NIH3T3 cells with and without Azacytidine treatment were analysed
Project description:HNF4α is a nuclear receptor regulating the transcription of genes involved mainly in development, cell differentiation and metabolism. Opposite functions for the two classes of P1 and P2 isoforms of HNF4α have recently been highlighted. These classes include 12 variants of HNF4α that can be expressed by the use of two promoters and by alternative splicing. Until now, the characterization of this transcription factor has ignored this diversity and has remained confined to the study of a fraction of the isoforms. We therefore wanted to clarify the situation by specifically characterizing the transcriptional functions of the 12 isoforms of HNF4α. We have generated for this purpose stable lines expressing each isoform of HNF4α in HCT 116 cells. We analyzed the whole transcriptome associated with each isoform by sequencing RNA, as well as their proteome by a BioID approach coupled to quantitative mass spectrometry. We noted major differences in the transcriptional function of the 12 isoforms. The α4, α5 and α6 isoforms have been characterized for the first time, and show a greatly reduced transcriptional potential. We have shown that these isoforms are unable to recognize the consensus response element of HNF4α. The α1 and α2 isoforms are the most potent regulators of gene expression, while the α3 isoform exhibits significantly reduced activity. Several transcription factors and coregulators have been identified as potential specific partners for certain HFH4α isoforms. The IRF-2BP2 co-repressor interacts specifically with isoforms which include the long form of the F domain of HNF4α. This specific interaction could explain the large number of genes modulated negatively by α1 and α2 compared to α3. The analysis integrating the vast amount of transcriptomic and proteomic data allows the identification of transcriptional regulatory mechanisms specific to certain isoforms, demonstrating the importance of considering all isoforms which can have diverse functions.
Project description:DNA microarray experiments were used to compare gene expression profiles of untreated and 5-azacytidine treated Escherichia coli at both logarithmic phase and early stationary phase The goal was to determine the effect of cytosine DNA methylation loss on gene expression (5-azacytidine is a methylation inhibitor) Overall design: Untreated versus 5-azacytidine-treated cells at logarithmic phase and early stationary phase; 5 biological replicates both all experiments
Project description:We developed genetically engineered Hepa/8F5 cells (available from RIKEN BioResource Center [RCB4661]), in which genes of eight liver-enriched transcription factors (LETFs)—hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β [FOXA2], HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ—were transduced into murine hepatoma Hepa1-6 cells as drug-inducible expression cassettes [Biochem. Eng. J., 60, 67–73 (2012)]. Hepa/8F5 cells can induce high liver functions by the addition of an inducer drug (doxycycline; Dox) via overexpression of LETF genes.
Project description:5-Azacytidine or 5-aza-2’-deoxycytidine is a chemical analogue of the cytosine nucleoside used in DNA and RNA. Cells in the presence of 5-azacytidine incorporate it into DNA during replication and RNA during transcription. The incorporation of 5-azacytidine into DNA or RNA inhibits methyltransferase thereby causing demethylation in that sequence, affecting the way that cell regulation proteins are able to bind to the DNA/RNA substrate. Inhibition of DNA occurs through the formation of stable complexes between the molecule and with DNA methyltransferases, thereby saturating the cells methylation machinery. 5-azacytidine is mainly used in the treatment of myelodysplastic syndrome. Changes in gene expression levels in Erbb2 transfected murine fibroblast cell line after treatment with Azacytidine in two different concentrations were analysed. Keywords: cDNA microarray, murine fibroblast, azacytidine treatment, carcinogenesis Gene expression levels of Erbb2 transfected NIH3T3 cell lines treated with 5µM and 10µM Azacytidine were compared to Erbb2 transfected but non-treated NIH3T3 cell line. For each Azacytidine concentration 4-8 experiments were performed including 50% dye swaps. As a controll experiments gene expression levels of NIH3T3 cells with and without Azacytidine treatment were analysed