Dataset Information

Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.

ABSTRACT: Using the rat insulinoma cell line INS-1 we generated beta-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the beta-cell specific phenotype of the Flp-In T-REx cell clones. Using a clone expressing the HNF6, HNF4alpha and HNF1beta transcription factors at a limited level, we introduced the expression vectors encoding these factors. We show efficient tetracycline induction of these transcription factors by western blots and immunocytochemistry. Microarrays reveal that these three factors affect a similar number of genes with only few genes regulated in common. Statistical analysis reveals that the three transcription factors affect genes categorized to different biological processes. Furthermore, we document the usefulness of these Flp-In T-REx cells for the functional analysis of mutated HNF1beta transcription factors found in human MODY5 patients. We show that the expression of the mutant P328L329del and A263insGG affects only very few transcripts and these are predominantly distinct from those induced by wild-type HNF1beta.

SUBMITTER: Thomas H

PROVIDER: S-EPMC528820 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.

Thomas Heike H Senkel Sabine S Erdmann Silke S Arndt Tanja T Turan Gülüzar G Klein-Hitpass Ludger L Ryffel Gerhart U GU

Nucleic acids research 20041101 19

Using the rat insulinoma cell line INS-1 we generated beta-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the ...[more]

PMID: 15520459

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Project description:BACKGROUND & AIMS:The Onecut 1 transcription factor (Oc1, a.k.a. HNF6) promotes differentiation of endocrine and duct cells of the pancreas; however, it has no known role in acinar cell differentiation. We sought to better understand the role of Oc1 in exocrine pancreas development and to identify its direct transcriptional targets. METHODS:Pancreata from Oc1Δpanc (Oc1fl/fl;Pdx1-Cre) mouse embryos and neonates were analyzed morphologically. High-throughput RNA-sequencing was performed on control and Oc1-deficient pancreas; chromatin immunoprecipitation sequencing was performed on wild-type embryonic mouse pancreata to identify direct Oc1 transcriptional targets. Immunofluorescence labeling was used to confirm the RNA-sequencing /chromatin immunoprecipitation sequencing results and to further investigate the effects of Oc1 loss on acinar cells. RESULTS:Loss of Oc1 from the developing pancreatic epithelium resulted in disrupted duct and acinar cell development. RNA-sequencing revealed decreased expression of acinar cell regulatory factors (Nr5a2, Ptf1a, Gata4, Mist1) and functional genes (Amylase, Cpa1, Prss1, Spink1) at embryonic day (e) 18.5 in Oc1Δpanc samples. Approximately 1000 of the altered genes were also identified as direct Oc1 targets by chromatin immunoprecipitation sequencing, including most of the previously noted genes. By immunolabeling, we confirmed that Amylase, Mist1, and GATA4 protein levels are significantly decreased by P2, and Spink1 protein levels were significantly reduced and mislocalized. The pancreatic duct regulatory factors Hnf1β and FoxA2 were also identified as direct Oc1 targets. CONCLUSIONS:These findings confirm that Oc1 is an important regulator of both duct and acinar cell development in the embryonic pancreas. Novel transcriptional targets of Oc1 have now been identified and provide clarity into the mechanisms of Oc1 transcriptional regulation in the developing exocrine pancreas. Oc1 can now be included in the gene-regulatory network of acinar cell regulatory genes. Oc1 regulates other acinar cell regulatory factors and acinar cell functional genes directly, and it can also regulate some acinar cell regulatory factors (eg, Mist1) indirectly. Oc1 therefore plays an important role in acinar cell development.

Dataset Information

Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.

Publications

Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.

Similar Datasets

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