ABSTRACT
Hepatocyte nuclear factor 4alpha (HNF4alpha) is an orphan receptor of the nuclear receptor superfamily and expressed in vertebrates as a tissue-specific transcription factor in liver, kidney, intestine, stomach, and pancreas. It also plays a crucial role in early embryonic development and has been identified as a maternal component in the Xenopus egg. We now report on an activity present in Xenopus embryos that inhibits the DNA binding of HNF4. This HNF4 inhibitor copurifies with a 25-kDa protein under nondenaturing conditions but can be separated from this protein by sodium dodecyl sulfate treatment. Protease treatment of the inhibitor results in a core fragment of about 5 kDa that retains full inhibitory activity. The activity of the HNF4 inhibitor can also be monitored in the absence of DNA, as it alters the mobility of the HNF4 protein in native polyacrylamide gels and the accessibility of antibodies. Comparing the activity of the HNF4 inhibitor with acyl coenzyme A's, recently proposed to be ligands of HNF4, we observe a more stringent specificity for the HNF4 inhibitor activity. Using deletion constructs of the HNF4 protein, we could show that the potential ligand-binding domain of HNF4 is not required, and thus the HNF4 inhibitor does not represent a classical ligand as defined for the nuclear receptor superfamily. Based on our previous finding that maternal HNF4 is abundantly present in Xenopus embryos but the target gene HNF1alpha is only marginally expressed, we propose that the HNF4 inhibitor functions in the embryo to restrict the activity of the maternal HNF4 proteins.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , Nuclear Proteins , Ovum/chemistry , Phosphoproteins/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Binding Sites , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Hepatocyte Nuclear Factor 4 , Ligands , Protein Binding/drug effects , RNA, Messenger, Stored , Transcription Factors/biosynthesis , Xenopus/embryology , Xenopus ProteinsABSTRACT
The transcription factor hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue specific transcription factor mainly expressed in the liver, kidney, intestine and the endocrine pancreas, but is also an essential regulator for early embryonic events. Based on its protein structure HNF4alpha is classified as an orphan member of the nuclear receptor superfamily. Comparing HNF4alpha transcription factors in the differentiated and dedifferentiated murine hepatocyte cell line MHSV-12 we identified in dedifferentiated cells the novel splice variant HNF4alpha7. This variant is characterized by an alternative first exon and has a lower transactivation potential in transient transfection assays using HNF4 dependent reporter genes. HNF4alpha7 mRNA and the corresponding protein are expressed in the undifferentiated pluripotent embryonal carcinoma cell line F9, whereas HNF4alpha1 only appears after differentiation of F9 cells to visceral endoderm. HNF4alpha7 mRNA is also found in totipotent embryonic stem cells. However, the function of HNF4alpha7 seems not to be restricted to embryonic cells as the HNF4alpha7 mRNA is also present in adult tissues, most notably the stomach. All these features suggest that the presence of distinct splice variants of HNF4alpha modulates the activity of HNF4alphain a cell type specific way.
Subject(s)
Alternative Splicing , DNA-Binding Proteins , Genetic Variation , Liver/chemistry , Phosphoproteins/analysis , Phosphoproteins/genetics , Transcription Factors/analysis , Transcription Factors/genetics , Animals , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cell Differentiation , Cell Line , Embryonal Carcinoma Stem Cells , Exons , Gene Expression , Glucocorticoids/pharmacology , Hepatocyte Nuclear Factor 4 , Mice , Molecular Sequence Data , Neoplastic Stem Cells/metabolism , Organ Specificity , RNA, Messenger/analysis , Sequence Alignment , Transcriptional Activation , TransfectionABSTRACT
In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.
