The core region of the Emicro3' transcriptional enhancer that drives the expression of the teleost IGH locus has been characterized functionally in two species, the catfish (Ictalurus punctatus) and the zebrafish (Danio rerio). These studies have suggested important differences: whereas the catfish enhancer acts through an E-box and two octamer motifs, the zebrafish enhancer exerts its major effects through two E-box motifs alone. In this study, the function of the catfish enhancer was reexamined in a broader comparative context within the teleosts. Electrophoretic mobility shift assays of motifs from catfish, zebrafish, and Fugu were conducted to determine their ability to bind catfish E-protein and Oct transcription factors. Transient expression assays were conducted using a region of the catfish core enhancer that includes a newly described hybrid octamer/E-box motif. Sequences homologous to the Emicro3' enhancer region from six teleosts were aligned to determine conserved regions ("phylogenetic footprinting"). These studies allowed the following conclusions to be drawn: (1) The important 3'E-box motif described in the zebrafish corresponds in the homologous region of the catfish enhancer to an Oct motif with a newly described negative regulatory function and (2) Comparison of the Emicro3' enhancer sequences of six teleosts indicates that while a variety of octamer and E-box motifs are found in this region, strict evolutionary conservation of the important functional elements of the teleost Emicro3' enhancer has not occurred.

Single genes orthologous to HEB and to E2-2 were identified. In contrast, two members of the E2A gene family were identified in Takifugu; one of these shows the alternative processing of transcripts that identifies it as the ortholog of the E12/E47-encoding mammalian E2A gene, whereas the second Takifugu E2A gene has no predicted alternative splice products. A novel fifth E protein gene (EX) was identified in Takifugu. Phylogenetic analysis revealed four E protein branches among vertebrates: EX, E2A, HEB, and E2-2. comparative immunology; phylogeny; gene structure; catfish TRANSCRIPTIONAL CONTROL of the IGH locus in a teleost fish differs greatly from that described in the mammals. In the channel catfish, Ictalurus punctatus, the major enhancer (E3Ј) lies between the and ␦ genes (11), and its function is dependent on transcription factors binding to two variant octamer motifs and to a single E5 site (4). The prototype E2 and E5 E-box motifs (consensus sequence CANNTG) were originally discovered within the mammalian immunoglobulin intronic enhancer (E) by A. Ephrussi, after whom they are named (2, 8). E proteins are class I basic helix-loop-helix (bHLH) transcription factors that are ubiquitously expressed but are nevertheless involved in many cell type-specific functions, including the development of the lymphoid system and the transcriptional control of many T-and B-cell-specific genes.The mammalian E protein family consists of three members: E2A (with alternative splice products E12 and E47), HEB, and E2-2 (6, 10). In mammals, these proteins play major roles in several essential lymphoid processes such as immunoglobulin and T-cell antigen receptor (TCR) gene rearrangement as well as the expression of activation-induced cytidine deaminase, which mediates somatic hypermutation and class-switch recombination (6, 16). The Id proteins, which are class V bHLH transcription factors, also play an important role as negative regulators of gene transcription driven by E proteins, since they can heterodimerize with E proteins to inhibit their binding to the DNA (13,19).In the channel catfish significant effort has been made to understand the nature of the Oct and E protein transcription factors through which the E3Ј enhancer operates (5, 9, 14, 15). In catfish B cells the major expressed E protein was shown to be an ortholog of HEB. This transcription factor is unique for a HEB factor in that it is expressed as two different isoforms (CFEB1 and 2) that are derived by alternative splicing of a primary transcript (9). Both isoforms are capable of binding to the E5 motif, forming homo-or heterodimers, and strongly driving transcription from the E3Ј core enhancer in a E5-dependent manner (9). Recently, catfish homologs of E2A have been discovered (AY770493 and AY860223). One of these, E2A1, has similar functional properties to CFEB, although it is expressed at lower levels (9a).Projects designed to investigate gene families, such as the E proteins of catfish, must always consider the question "How do we know that we have iden...

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Background: The enhancer (Eμ3') of the immunoglobulin heavy chain locus (IGH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding μE5 site. An orthologue to the Oct2 transcription factor has previously been cloned in catfish and is a functionally active transcription factor. This study was undertaken to clone and characterize the Oct1 transcription factor, which has also been shown to be important in driving immunoglobulin gene transcription in mammals.

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