Function of E-protein dimers expressed in catfish lymphocytes

Abstract

E-proteins are essential class I bHLH transcription factors that play a role in lymphocyte development. In catfish lymphocytes the predominant E-proteins expressed are CFEB (a homologue of HEB) and E2A1, which both strongly drive transcription. In this study the role of homodimerization versus heterodimerization in the function of these catfish E-proteins was addressed through the use of expression constructs encoding forced dimers. Constructs expressing homo- and heterodimers were transfected into catfish B cells and shown to drive transcription from the catfish IGH enhancer. Expression from an artificial promoter containing a trimer of muE5 motifs clearly demonstrated that the homodimer of E2A1 drove transcription more strongly (by a factor of 10-25) than the CFEB homodimer in catfish B and T cells, while the heterodimer showed intermediate levels of transcriptional activation. Both CFEB1 and E2A1 proteins dimerized in vitro, and the heterodimer CFEB1-E2A1 was shown to bind the canonical muE5 motif.

Figure 1. Heterodimers of CFEB1-E2A1 drive transcription from the core region of the IGH enhancer in catfish B cells

(A) Schematic of expression constructs for the forced dimerization (including the linker sequence) of catfish E-proteins. (B) Transcriptional activity driven from the reporter constructs by cotransfection of forced dimer expression vectors (pRc/ELE, pRc/CLC, and pRc/CLE) in the catfish B cell line (1G8). The p-values (below B) were calculated by student’s T-test (n=7). The structure of the reporter construct (pGL/Δ56/R#2) is shown at the bottom of the figure. Abbreviations: H; Hind III, S; Sac II, A/N; Apa I or Not I, E; E2A1, C; CFEB1, ELE; E2A1-Linker-E2A1, CLC; CFEB1-Linker-CFEB1, CLE; CFEB1-Linker-E2A1.

Figure 2. Heterodimers of CFEB1-E2A1 drive transcription from a μE5-dependent reporter construct in catfish lymphoid cell lines

Transcriptional activities in catfish 1G8 B cells (A) and G14D T cells (B) are shown. The p-values calculated by student’s T-test (n=6) are indicated below the figures. The structure of the reporter construct containing the μE5 trimer artificial enhancer (pGL/Δ56/μE5x3) is shown below.

Figure 3. Heterodimers of CFEB1-E2A1 bind μE5 motifs

(A) EMSA assessing the binding of in vitro produced recombinant CFEB1-E2A1 protein to μE5 motifs. (B) The molecular size of the forced E-protein dimers was confirmed by SDS-PAGE. The ³⁵S-met labeled E2A1 monomer, CFEB1 monomer, and CFEB1-E2A1 dimer were produced in the TNT system. Molecular size markers (kDa) are shown to the left of the panels. The monomeric and dimeric shifts are indicated by black arrows.

Figure 4. The E2A1 protein can form homo- and heterodimers

(A) Dimerization was assayed in pull-down experiments by affinity chromatography on S-protein agarose. In vitro synthesized ³⁵S-labeled E2A1 was mixed with in vitro synthesized unlabeled Stagged E2A1, CFEB1 or CFEB2 pre-bound to S-protein agarose. Lanes 1, 4, and 7 show the input (1/100 volume) of ³⁵S-labeled E2A1 protein. Lane 3 shows the homodimerization of E2A1. The heterodimerization of E2A1 with CFEB1 or CFEB2 is shown in lanes 6 and 9. (B) Western blot analysis of the in vitro synthesized S-tagged E2A1, CFEB1 and CFEB2, developed using S-protein conjugated with HRP. The abbreviations E, C1, C2 and S indicate E2A1, CFEB1, CFEB2, and S-tag peptide, respectively.