ABSTRACT
The majority of human pancreatic adenocarcinomas display a ductal phenotype; experimental studies indicate that tumors with this phenotype can arise from both acinar and ductal cells. In normal pancreas acinar cells, the pancreas transcription factor 1 transcriptional complex is required for gene expression. Pancreas transcription factor 1 is a heterooligomer of pancreas-specific (p48) and ubiquitous (p75/E2A and p64/HEB) basic helix-loop-helix proteins. We have examined the role of p48 in the phenotype of azaserine-induced rat DSL6 tumors and cancers of the human exocrine pancreas. Serially transplanted acinar DSL6 tumors express p48 whereas DSL6-derived cell lines, and the tumors induced by them, display a ductal phenotype and lack p48. In human pancreas cancer cell lines and tissues, p48 is present in acinar tumors but not in ductal tumors. Transfection of ductal pancreas cancers with p48 cDNA did not activate the expression of amylase nor a reporter gene under the control of the rat elastase promoter. In some cell lines, p48 was detected in the nucleus whereas in others it was cytoplasmic, as in one human acinar tumor. Together with prior work, our findings indicate that p48 is associated with the acinar phenotype of exocrine pancreas cancers and it is necessary, but not sufficient, for the expression of the acinar phenotype.
Subject(s)
Adenocarcinoma/genetics , Cell Differentiation/genetics , Helix-Loop-Helix Motifs/genetics , Pancreas/pathology , Pancreatic Neoplasms/genetics , Transcription Factors/genetics , Adenocarcinoma/pathology , Amino Acid Sequence , Animals , Antimetabolites, Antineoplastic/pharmacology , Azaserine/pharmacology , Disease Models, Animal , Humans , Molecular Sequence Data , Pancreatic Neoplasms/pathology , Phenotype , RNA, Messenger/analysis , Rats , Tumor Cells, CulturedABSTRACT
Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism.
Subject(s)
Avian Proteins , DNA-Binding Proteins/metabolism , Histones/genetics , Regulatory Sequences, Nucleic Acid , Transcription Factors/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Chickens , DNA Primers , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , Erythrocytes/metabolism , Female , Glycosylation , HeLa Cells , Histones/biosynthesis , Humans , Kinetics , Molecular Sequence Data , Mutagenesis , Phosphorylation , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Deletion , Transcription Factors/biosynthesis , Transcription Factors/chemistry , TransfectionABSTRACT
Expression of histone H5, like that of other erythrocyte specific proteins, declines during the latter stages of erythroid maturation because of a decrease in the rate of gene transcription. Here, we report the isolation of cIBR (chicken initiation binding repressor), a 75 kDa DNA binding glycoprotein from mature chicken erythrocytes that recognizes sequences spanning the transcription start sites of the H5 gene. cIBR was found to repress transcription from the H5 promoter in vitro and this effect could be relieved by mutations that lowered the affinity of the factor for its cognate sequence. cIBR inhibited transcription by interfering with assembly of the initiation complex, but it did not affect transcription from pre-assembled complexes. Consistent with this, binding of bacterially expressed human TFIID to the TATA element prevented subsequent binding of cIBR, although the opposite was not true. This, and the fact that cIBR had no effect when bound in a location upstream from the promoter, suggests that binding of cIBR to the start site region causes repression by direct interference with general transcription factors other than TFIID, possibly TFIIB. cIBR was found in mature and relatively late erythrocytes but not in early erythroid cells which actively transcribe the H5 gene; the transcriptionally active cells contain instead cIBF (chicken initiation binding factor). Purified cIBF is a non-glycosylated 68-70 kDa DNA binding protein(s) which also recognizes the region of transcription initiation of the H5 gene.
