The immunoglobulin heavy chain VH6-1 promoter regulates Ig transcription in non-B cells

Abstract

Background: Non-B cell immunoglobulins (Igs) are widely expressed in epithelial cancer cells. The past 20 years of research have demonstrated that non-B cell Igs are associated with cancer cell proliferation, the cellular cytoskeleton and cancer stem cells. In this study we explored the transcriptional mechanism of IgM production in non-B cells.

Methods: The promoter region of a V-segment of the heavy mu chain gene (VH6-1) was cloned from a colon cancer cell line HT-29. Next, the promoter activities in non-B cells and B-cells were detected using the dual-luciferase reporter assay. Then the transcription factor binding to the promoter regions was evaluated by electrophoretic mobility shift assays (EMSAs) and gel supershift experiments.

Results: Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells. No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1. In addition, Oct-1 was found to bind to the octamer element of the Ig gene promoter in cancer cells, in contrast to B cells, which utilize the transcriptional factor Oct-2.

Conclusion: The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.

Keywords: Non-B cells; Oct-1; Promoter activity; Transcriptional regulation; VH6-1.

Figure 1

The 5′-flanking sequence of VH6-1 exhibits promoter activity in non-B cancer cells. (A) Schematic diagram of 5′-flanking 1.2-kb pGL3 construct. (B) The 1.2-kb fragments amplified from upstream of VH6-1 in HT-29 cells by PCR. (C) The 1.2-kb pGL3 construct was transfected into HeLa, HeLa MR, HeLa S3, HT-29, MDA-MB-231, HEK 293,L02, HK2, Raji or Jurkat cells. Luciferase activity was measured using a dual-luciferase reporter system. The results are representative of three independent experiments after normalization to renilla luciferase activity (internal controls). Each bar represents mean ± SD.

Figure 2

The octamer element is important but not essential for non-B cell-derived Ig gene transcription. (A) Schematic diagram of mutated 5′ deletion truncations of the 1.2-kb pGL3 construct with a 4-bp deletion in the octamer motif (AGGCAAAT). (B) The intact and mutated 5′ deletion truncations of the 1.2-kb pGL3 construct were transfected into HeLa, HeLa MR, HeLa S3, HT-29, MDA-MB-231, HEK 293, L02, HK2, and Raji cells. Luciferase activity was measured using a dual-luciferase reporter system. The results are representative of three independent experiments after normalization to renilla luciferase activity (internal controls). Each bar represents mean ± SD.

Figure 3

The sequence 1200 bp to 300 bp upstream of VH6-1 contains no regulatory elements. (A) Schematic diagram of 5′-flanking 300-bp pGL3 construct. (B) The 300-bp deletion fragment amplified from the 1.2-kb fragment containing the VH6-1 promoter by PCR. (C) The 300-bp deletion truncations of the 1.2-kb pGL3 construct exhibited similar promoter activity to the 1.2-kb pGL3 construct in HT-29 and Raji cells.

Figure 4

Oct-1 but not Oct-2 binds to the octamer element. (A) Oct-2 was not detected in epithelial cancer cells by RT-PCR. (B) The EMSA assay for octamer motif binding factors located in the promoter region of VH6-1 in HT-29 cells. The 40-bp DNA fragment was derived from upstream of the VH6-1 gene and contains the octamer motif, while the 32-bp DNA fragment was derived from the 40-bp DNA fragment with an 8-bp deletion in the octamer motif. (C) The super-shift assay for octamer motif binding factors with the addition of an anti-Oct-1 or anti-Oct-2 antibody in the binding reaction system. The results are representative of three independent experiments. EMSA, electrophoretic mobility shift assay. (D) The Oct-1 binding DNA fragment of the VH6-1 promoter was amplified via Chip-related PCR. Negative control: no template in the PCR reaction system; positive control: the sonicated chromatin fragments of the cells were used as the PCR template; control group: no antibody added to the IP system; test group: dilutions of IP were used as templates for PCR to amplify the Oct-1 binding DNA sequence. The results are representative of three independent experiments.