Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4alpha and HNF1beta in a pancreatic beta-cell line.
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.
Thomas
Heike
H
Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, D-45122 Essen, Germany.
Senkel
Sabine
S
Erdmann
Silke
S
Arndt
Tanja
T
Turan
Gülüzar
G
Klein-Hitpass
Ludger
L
Ryffel
Gerhart U
GU
D000818:Animals; D045744:Cell Line, Tumor; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D051539:Hepatocyte Nuclear Factor 1-beta; D051557:Hepatocyte Nuclear Factor 4; D051559:Hepatocyte Nuclear Factor 6; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C496495:HNF1B protein, human; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D051559:Hepatocyte Nuclear Factor 6; C496594:Hnf4a protein, rat; D018398:Homeodomain Proteins; C496605:ONECUT1 protein, human; C496607:Onecut1 protein, rat; D010750:Phosphoproteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D051539:Hepatocyte Nuclear Factor 1-beta; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D013752:Tetracycline