ABSTRACT
The transcriptional enhancer (Emu3') of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single muE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a muE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Emu3') in a manner dependent on the presence of the muE5 site. Both CFEB1 and -2 bound the muE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Evolution, Molecular , Ictaluridae/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Sequence Homology, Nucleic Acid , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription, Genetic , Alternative Splicing , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Binding Sites/genetics , Cell Line , Cell Line, Transformed , Cloning, Molecular , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Enhancer Elements, Genetic , Genetic Markers/immunology , Helix-Loop-Helix Motifs , Ictaluridae/genetics , Immunoglobulin mu-Chains/genetics , Immunoglobulin mu-Chains/metabolism , Molecular Sequence Data , Organ Specificity/genetics , Organ Specificity/immunology , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , RNA Precursors/biosynthesis , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Transcription Factors/metabolism , Transcription Factors/physiology , Transcription, Genetic/immunologyABSTRACT
Oct2 transcription factors of the catfish (Ictalurus punctatus) are expressed as alternatively spliced alpha and beta isoforms. Functional analysis revealed an N-terminal glutamine (Q)-rich transactivation domain common to both isoforms of catfish Oct2. A C-terminal proline, serine, threonine (PST)-rich activation domain was identified exclusively in the beta isoform. Activation domains of fish and mammalian Oct2 showed cell type- and species-specific activity correlated with their biochemical composition (Q-rich vs PST-rich). In contrast the activation domains of the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator of fish and mammals showed no correlation of activity with biochemical composition or species of origin. Although isolated catfish Oct2 activation domains were unable to drive transcription from a site 1.9kb distal to the promoter, Oct2beta activated transcription from both an IgH enhancer and an array of octamer motifs at this distal position. The properties of catfish Oct2 activation domains differ depending on whether they are studied in isolation or as components of the intact transcription factor.
