Interaction between E-protein and Oct transcription factors in the function of the catfish IGH enhancer

Abstract

Transcriptional control of the immunoglobulin heavy chain (IGH) locus in the channel catfish, Ictalurus punctatus, is incompletely understood. It is, however, known that 2 variant octamer motifs and a microE5 motif in the core region of the enhancer (Emicro3') are important in driving transcription, and it has been suggested that interaction between transcription factors (Oct factors and E-proteins) bound to these sites contributes to enhancer function. In this study, the functional relationships between the microE5 motif, the proximal octamer motif, and the factors that bind them have been examined. The results of mutational analysis of these motifs showed that their interaction is important to driving transcription from the enhancer. Furthermore, the catfish Oct transcription factors were capable of a physical interaction with the catfish E-proteins. These results support a role for interaction between transcription factors bound to the octamer and microE5 motifs in the function of the Emicro3' enhancer.

Figure 1. Effect of mutational inactivation of octamer and μE5 motifs in the Eμ3′ enhancer

(A) The Oct#11 and μE5 motifs were inactivated singly or together by site-directed mutation. A schematic illustrating the motifs mutated in the core region (R#2) of the Eμ3′ enhancer is shown on the left. The oval, rectangle, and diamond in the schematic illustrations indicate the motifs to be bound with transcription factors: Oct, E-protein, and an unknown protein (X; Hikima et al, 2006), respectively. Covered black crosses indicate a mutated motif. Transcriptional activity of the mutated reporter constructs, shown as percent activity relative to the wild type (WT) R#2, upon transient transfection into catfish 1G8 B cells. The standard error (SE) is denoted by the error bars, and the asterisk “*” indicates the significant difference between each of these constructs at p < 0.01, as determined by two-tailed Student's t-test (n=9). (B) Schematic showing potential interactions between transcription factors and calculations for the contributions of the Oct and μE5 motifs derived from the results in (A). Abbreviations: O#10, octamer#10; O#11, octamer#11.

Figure 2. Interaction between Oct transcription factors and E-proteins in the function of the Eμ3′ enhancer

(A) Transcriptional activation of the Eμ3′ enhancer: effect of changing the distance between the octamer and μE5 motifs. The distance between Oct#11 and μE5 site was increased or decreased as indicated in the sequences of O/E+20bp, O/E+14bp, and O/E-5bp. (B) Schematic illustrating the orientation of the motifs on the same or opposite faces of the DNA helix and the structures of luciferase (LUC) reporter vectors are shown on the left side of graph. Transcriptional activity of mutated reporter constructs, shown as fold activation, upon transient transfection into catfish 1G8 cells. The orientation (direction) of box at μE5 site indicates the actual face of DNA motif to bind with an E-protein. The standard error (SE) is indicated with the error bars, and the number of replicates is indicated in parentheses to the right. The asterisk “*” indicates the significant difference between each of experimental results at p > 0.01, as determined by two-tailed Student's t-test. (C) The results from B plotted as a function of the separation (bp) between the Oct#11 and μE5 motifs. Abbreviations: WT; wild type, O/E; octamer #11 and μE5. (D) Interaction was assayed in pull-down experiments by affinity chromatography on S-protein agarose. In vitro synthesized ³⁵S-labeled catfish Oct transcription factors (Oct2α, Oct2β, or Oct1) were mixed with in vitro synthesized non-labeled S-tagged catfish E-proteins (CFEB1, CFEB2, or E2A1) pre-bound to S-protein agarose beads. Lanes labeled “Input (1/50)” show 2% of the starting material (³⁵S-labeled Oct transcription factors) entered into the reaction. That the interactions detected in this assay are attributable to the catfish E-proteins and not to the epitope tag is shown by the lack of interaction when the S-tag peptide alone was used (lanes labeled “S-peptide”). (E) Control confirming S-tag function using the in vitro synthesized ³⁵S-labeled and S-tagged proteins (CFEB1, CFEB2, E2A1), captured by S-protein chromatography and separated on 7% SDS-PAGE.