Characterization of an Oct1 orthologue in the channel catfish, Ictalurus punctatus: a negative regulator of immunoglobulin gene transcription?

Abstract

Background: The enhancer (Emu3') 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 muE5 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.

Results: An orthologue of Oct1, a POU family transcription factor, was cloned from a catfish macrophage cDNA library. The inferred amino acid sequence of the catfish Oct1, when aligned with other vertebrate Oct1 sequences, revealed clear conservation of structure, with the POU specific subdomain of catfish Oct1 showing 96% identity to that of mouse Oct1. Expression of Oct1 was observed in clonal T and B cell lines and in all tissues examined. Catfish Oct1, when transfected into both mammalian (mouse) and catfish B cell lines, unexpectedly failed to drive transcription from three different octamer-containing reporter constructs. These contained a trimer of octamer motifs, a fish VH promoter, and the core region of the catfish Emu3' IGH enhancer, respectively. This failure of catfish Oct1 to drive transcription was not rescued by human BOB.1, a co-activator of Oct transcription factors that stimulates transcription driven by catfish Oct2. When co-transfected with catfish Oct2, Oct1 reduced Oct2 driven transcriptional activation. Electrophoretic mobility shift assays showed that catfish Oct1 (native or expressed in vitro) bound both consensus and variant octamer motifs. Putative N- and C-terminal activation domains of Oct1, when fused to a Gal4 DNA binding domain and co-transfected with Gal4-dependent reporter constructs were transcriptionally inactive, which may be due in part to a lack of residues associated with activation domain function.

Conclusion: An orthologue to mammalian Oct1 has been found in the catfish. It is similar to mammalian Oct1 in structure and expression. However, these results indicate that the physiological functions of catfish Oct1 differ from those of mammalian Oct1 and include negative regulation of transcription.

Figure 1

An Oct1 orthologue in catfish. A. Megalin (DNAStar) amino acid alignment of Oct1 proteins. Conserved residues are shaded by grey boxes. The boxed portion indicates the bipartite POU DNA binding domain. Glutamine rich regions are denoted by arrows and text Gln1 or Gln2. Serine and thrionine rich residues are denoted by arrows and S/T rich. The linker region is also designated by text. Gaps in the sequence alignment are indicated by a dash. B. Schematic comparing the domain structure of Oct transcription factors. N- and C- terminal domains are shown as checkered boxes, POU domains are shown hatched with diagonal lines, and the linker region is shown as a black box. The percent identity values (derived using the Megalign program, DNAStar) are based on the amino acid sequences and are shown below the schematic. C. Neighbor joining phylogenetic tree [49] of Oct1 and Oct2 transcription factor sequences (see Methods). The C. elegans homeobox gene was used as the outgroup. The three branches for Oct1, Oct2 and the invertebrate Oct sequences are indicated by brackets, and the bootstrap value for each node is shown.

Figure 2

Catfish Oct1 is widely expressed. RNA was isolated from the catfish T cell line (G14D) and B cell line (1G8) as well as catfish tissues (spleen, head kidney, trunk kidney, brain, heart, gill, and intestine) and subjected to RT-PCR analysis to detect Oct1 (top panel), as described in the Methods section. RT-PCR detection of the catfish actin transcript is shown in the bottom panel.

Figure 3

Catfish Oct1 fails to drive transcription from an octamer-dependent reporter construct. A schematic representation of the reporter construct (pO3-Δ56-CAT [31]) containing an octamer trimer is shown in A. The octamer motifs are shown upstream of the TATA box (from the minimal c-fos promoter) and the CAT reporter gene is shown as an open box. Transcriptional activation (as fold increase) was measured following co-transfection into the catfish B cell line (1G8) of the reporter construct and an Oct expression construct. A. Transcriptional activation driven by catfish Oct2β (pRc/CMV/Oct2β) or catfish Oct1 (pRc/CMV/Oct1). B. Transcriptional activation driven by catfish Oct2β (pRc/CMV/Oct2β), catfish Oct1 (pRc/CMV/Oct1) and human BOB.1 (pRc/CMV/hBOB.1), or by a co-transfection of catfish Oct2β with human BOB.1, or catfish Oct1 with human BOB.1. C. Negative transcriptional regulation by catfish Oct1. One pM of catfish Oct2β expression construct was co-transfected with increasing amounts of catfish Oct1 (0.5, 1.0, 2.0 pM respectively) and the transcriptional activation of the reporter construct measured. Values for five replicate transfections in A and four replicate transfections in B and C are shown as mean ± SEM. Statistical significance was calculated by the Student T test assuming unequal variances. One asterisk indicates a p value of less than .05 and two asterisks indicates a p value of less than .01.

Figure 4

Catfish Oct1 fails to drive transcription from physiologically relevant reporter constructs. A. Schematic diagram of the reporter construct pGL3/Δ56/R#2 [50]. This contains the core of the catfish enhancer in which there are 2 octamer motifs (ovals) and a μE5 site (square) upstream of a minimal TATA box (circle) followed by the luciferase gene (open rectangle). This reporter construct was used in the experiments shown in panels A&B. A. Transcriptional activation driven from the core enhancer by catfish Oct2β (pRc/CMV/Oct2β) or catfish Oct1 (pRc/CMV/Oct1) in the mouse B cell line (J558L). B. Transcriptional activation in the catfish B cell line 1G8 driven from the core enhancer construct by catfish Oct2β (pRc/CMV/Oct2β), catfish Oct1 (pRc/CMV/Oct1), human BOB.1 (pRc/CMV/hBOB.1), or by a co-transfection of catfish Oct2β with human BOB.1, or catfish Oct1 with human BOB.1. C. (Top) Schematic diagram of the reporter construct (pFVH-CAT) that contains a full fish V_H promoter (arrow) followed by the CAT gene [27]. C. (Bottom) Transcriptional activation driven from the V_H promoter (pFVH-CAT) by catfish Oct2β (pRc/CMV/Oct2β) or catfish Oct1 (pRc/CMV/Oct1) co-transfected with the reporter construct into catfish 1G8 B cells. Values are presented as mean ± SD for 12 replicates (A), 9 replicates (B), and 4 replicates (C). Statistical significance was calculated by the Student T test assuming unequal variances. One asterisk indicates a p value of less than .05 and two asterisks indicate a p value of less than .01.

Figure 5

In vitro transcribed Oct transcription factors bind the octamer motif. A. EMSA demonstrating the binding of catfish Oct1 to the first octamer motif within the core enhancer. Lane 1, free probe. Lane 2, TNT reaction without template DNA (control). Lane 3, catfish Oct1 TNT product. Lane 4, catfish Oct1 TNT product + cold competitor. Lane 5, catfish Oct1 TNT product + scrambled competitor. Lane 6, catfish Oct1 TNT product + anti-Oct1 rabbit serum. Lane 7, catfish Oct1 TNT product + NRS (anti-Oct1 rabbit pre-bleed serum). Arrow depicts the Oct1 shift. B. EMSA demonstrating the binding of catfish Oct2β to the first octamer motif within the core enhancer. Lane 1, catfish Oct2β TNT product. Lane 2, catfish Oct2β TNT product + cold competitor. Lane 3, catfish Oct2β TNT product + scrambled competitor. Lane 4, catfish Oct2β TNT product + anti-Oct2β rabbit serum. Lane 5, catfish Oct2β TNT product + NRS (anti-Oct1 rabbit pre-bleed serum. C. Detection of ³⁵S-labeled TNT transcribed and translated Oct1 and Oct2β by SDS-PAGE and phosphor imaging. Solid black bands on the left hand side represent the kaleidoscope protein marker (Bio-Rad, Hercules, CA.), 222kDa, 129kDa, 83kDa, 43kDa (from top to bottom).

Figure 6

Endogenous Oct transcription factors bind the octamer motif. EMSA with nuclear extracts of the catfish B cell line 1G8. Lane 1, nuclear extract. Lane 2, nuclear extract + cold competitor. Lane 3, nuclear extract + scrambled competitor. Lane 4, nuclear extract + anti-Oct2 rabbit serum. Lane 5, nuclear extract + NRS (anti-Oct1 rabbit pre-bleed serum. Lane 6, nuclear extract + anti-Oct1 rabbit serum. Lane 7, nuclear extract + anti-Oct1 rabbit serum + anti-Oct2 rabbit serum. Oct1 and Oct2 shifts are designated by brackets. A lower bracket designates non specific binding labeled NS.

Figure 7

Schematic of Gal4 fusion proteins. A. Depicts the pG5/CAT reporter construct. Gal4 binding sites (grey hexagons), TATA promoter (grey circle) and the CAT gene (open rectangle). A schematic of the effector constructs used in B. CMV promoter (black arrow), Gal4 DNA binding domain (black rectangle), Oct1 POU domain (grey rectangle), Oct1 N-terminus (plaid rectangle), Oct1 C-terminus (black with white dotted rectangle), VP16 (diagonal stripped rectangle), Oct2β N-terminus (black diamond rectangle), Oct2β C-terminus (white with black dotted rectangle), Nucleolin (tire track rectangle), empty activation domain (dotted line).