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

Wu L, Liu Y, Zhu X, Zhang L, Chen J, Zhang H, Hao P, Zhang S, Huang J, Zheng J, Zhang Y, Zhang Y, Qiu X - Cancer Cell Int. (2014)

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

View Article: PubMed Central - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Central Laboratory, Peking University Cancer Hospital & Institute, Beijing, 100142 China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig4: 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.

Mentions: It is known that Oct-2 binds to the octamer element in mature B lymphocytes to initiate the expression of Ig. The Oct-2 transcript was not detected by RT-PCR in HeLa S3, HT-29 and Jurkat cells, compared with Daudi cells as a positive control (Figure 4A). Next, EMSAs were performed to evaluate the binding of TFs to the octamer element of the Ig VH6-1 gene in epithelial cancer cells. Nuclear protein fractions from HT-29 cells interacted with octamer elements of the VH6-1 upstream region, appearing in a non-denaturing electrophoresis hysteresis band. Compared with the 40-bp double-stranded oligonucleotides containing the octamer motif, the hysteresis band disappeared for the 32-bp oligonucleotides that did not contain an octamer motif, indicating that this interaction was specific (Figure 4B). The anti-Oct-1 antibody strongly super-shifted the binding complex of the HT-29 nuclear proteins, which indicated that no hysteresis band was detected because the DNA binding to the antibody-protein complex did not exit through holes (Figure 4C). These results also indicated that Oct-1 interacts with the octamer element to promote Ig VH6-1 expression in HT-29 cells.Figure 4


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

Wu L, Liu Y, Zhu X, Zhang L, Chen J, Zhang H, Hao P, Zhang S, Huang J, Zheng J, Zhang Y, Zhang Y, Qiu X - Cancer Cell Int. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4260249&req=5

Fig4: 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.
Mentions: It is known that Oct-2 binds to the octamer element in mature B lymphocytes to initiate the expression of Ig. The Oct-2 transcript was not detected by RT-PCR in HeLa S3, HT-29 and Jurkat cells, compared with Daudi cells as a positive control (Figure 4A). Next, EMSAs were performed to evaluate the binding of TFs to the octamer element of the Ig VH6-1 gene in epithelial cancer cells. Nuclear protein fractions from HT-29 cells interacted with octamer elements of the VH6-1 upstream region, appearing in a non-denaturing electrophoresis hysteresis band. Compared with the 40-bp double-stranded oligonucleotides containing the octamer motif, the hysteresis band disappeared for the 32-bp oligonucleotides that did not contain an octamer motif, indicating that this interaction was specific (Figure 4B). The anti-Oct-1 antibody strongly super-shifted the binding complex of the HT-29 nuclear proteins, which indicated that no hysteresis band was detected because the DNA binding to the antibody-protein complex did not exit through holes (Figure 4C). These results also indicated that Oct-1 interacts with the octamer element to promote Ig VH6-1 expression in HT-29 cells.Figure 4

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

View Article: PubMed Central - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Central Laboratory, Peking University Cancer Hospital & Institute, Beijing, 100142 China.

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.

No MeSH data available.


Related in: MedlinePlus