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. 2017 Jul 11;8(28):45105-45116.
doi: 10.18632/oncotarget.15126.

Egr-1 and RNA POL II Facilitate Glioma Cell GDNF Transcription Induced by Histone Hyperacetylation in Promoter II

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Free PMC article

Egr-1 and RNA POL II Facilitate Glioma Cell GDNF Transcription Induced by Histone Hyperacetylation in Promoter II

Bao-Le Zhang et al. Oncotarget. .
Free PMC article

Abstract

The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.

Keywords: Egr-1; GDNF; RNA POL II; glioma; histone acetylation.

Conflict of interest statement

CONFLICTS OF INTEREST

There are no conflicts of interest.

Figures

Figure 1
Figure 1. H3K9 acetylation of GDNF gene promoter region II in human high-grade glioma tissue (H), low-grade glioma tissue (L), and normal brain tissue (N)
(A) Schematic showing the Egr-1 binding site in human GDNF gene promoter region II. The black and white arrows indicate the ChIP-PCR primer positions. The transcription start site (TSS) of the promoter II-derived transcript is +1. Phylogenetic conservation values are indicated. (B) ChIP analyses with anti-acetyl-histone H3 (Lys9) antibody or normal rabbit IgG (negative control). Immunoprecipitated and input DNA were assayed using PCR with specific primers (P(−98/+56), P(−864/−793)). (C) The ChIP DNA value was standardized with input DNA, and the data are the percentages of input chromatin. *P < 0.05, **P < 0.01, ##P < 0.01.
Figure 2
Figure 2. Egr-1 binding to GDNF gene promoter region II in human high-grade glioma tissue (H), low-grade glioma tissue (L), and normal brain tissue (N)
(A) An anti-Egr-1 antibody or normal rabbit IgG was used for ChIP analysis following cross-linking and chromatin breakage. Specific primers for target P(−98/+56) and off-target P(−864/−793) areas and immunoprecipitated DNA and input DNA were detected with PCR. Specific primers for target area P(−98/+56) amplified target fragments to variable degrees, but using the same ChIP DNA template, the primer for the off-target area P(−864/−793) could not produce target fragments with PCR amplification, while target fragments were procured using input DNA. (B) After real-time PCR, ChIP DNA values were standardized with input DNA, and data are the percentages of chromatin. **P < 0.01.
Figure 3
Figure 3. RNA POL II recruitment to Egr-1 binding sites of GDNF promoter II in glioma tissues and cells
(A, B) RNA POL II recruitment was measured in different grades of glioma tissues using ChIP-PCR. (C, D) Recruitment was measured in rat C6 astroglioma cells and normal astrocytes using ChIP-PCR. **P < 0.01.
Figure 4
Figure 4. Egr-1 expression levels in glioma tissues and cells
(A, B) Relative expression of Egr-1 mRNA and protein in high-grade glioma tissue (H), low-grade glioma tissue (L) and normal brain tissue (N). (C, D) Relative Egr-1 mRNA and protein expression in rat C6 astroglioma cells and rat normal astrocytes (NA). β-actin was used as the internal reference protein. **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. Egr-1 overexpression significantly increased Egr-1 binding to GDNF promoter II, RNA POL II recruitment, and GDNF transcription in rat C6 glioma cells
C6 glioma cells in the logarithmic growth phase were inoculated into 6-well plates and then infected with 100 MOI of lentivirus Egr1-OE and lentivirus NC-EGFP when the cells reached 70% confluence. After 72 h, the virus infection rate was observed under the fluorescence microscope. (A, B) More than 90% of C6 glioma cells had normal morphology and showed green fluorescence. Real-time PCR, western blot, and ChIP-PCR showed that (C) Egr-1 mRNA and (D) protein expression significantly increased in C6 cells infected with lentivirus Egr1-OE (P < 0.01). (E) Egr-1 binding to GDNF promoter II, (F) RNA POL II recruitment in that region, and (G) GDNF mRNA expression were also increased significantly (P < 0.05). The results are from three independent experiments. *P < 0.05, **P < 0.01. The scale is 100 μm.
Figure 6
Figure 6. Histone hypoacetylation inhibited GDNF transcription induced by Egr-1 overexpression in GDNF promoter II in C6 glioma cells
Cells in the logarithmic growth phase were inoculated into 6-well plates and treated with 50 μM curcumin or 0.25% DMSO for 24 h when the cells reached 50% confluence. The cells were then infected with 100 MOI of lentivirus Egr 1-OE for 72 h. (A) H3K9 acetylation of the Egr-1 binding sites of GDNF promoter II in C6 glioma cells was measured using ChIP-PCR. (B) Egr-1 binding was measured using ChIP-PCR. (C) RNA POL II recruitment in the Egr-1 binding sites of GDNF promoter II in C6 glioma cells was measured using ChIP-PCR. (D) Relative GDNF mRNA expression was measured by real-time PCR. *P < 0.05, **P < 0.01, ###P < 0.001.
Figure 7
Figure 7. Co-existence but not binding of Egr-1 and RNA POL II in C6 glioma cell nuclei
(A) Egr-1 and RNA POL II protein locations in C6 glioma cells were determined by immunofluorescence. They were co-expressed in the nuclei of C6 cells with local overlapping regions. The scale is 100 μm. (B, C) The binding of Egr-1 to RNA POL II in C6 glioma cell nuclei was measured by co-IP with an Egr-1 antibody followed by western blotting with an RNA polymerase II antibody. Next, the reverse protocol was carried out. The results showed that the proteins were not bound in the nuclei of C6 glioma cells.

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References

    1. Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science. 1993;260:1130–32. - PubMed
    1. Grimm L, Holinski-Feder E, Teodoridis J, Scheffer B, Schindelhauer D, Meitinger T, Ueffing M. Analysis of the human GDNF gene reveals an inducible promoter, three exons, a triplet repeat within the 3′-UTR and alternative splice products. Hum Mol Genet. 1998;7:1873–86. - PubMed
    1. Baecker PA, Lee WH, Verity AN, Eglen RM, Johnson RM. Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene. Brain Res Mol Brain Res. 1999;69:209–22. - PubMed
    1. Shabtay-Orbach A, Amit M, Binenbaum Y, Na'ara S, Gil Z. Paracrine regulation of glioma cells invasion by astrocytes is mediated by glial-derived neurotrophic factor. Int J Cancer. 2015;137:1012–20. - PubMed
    1. Qu DW, Liu Y, Wang L, Xiong Y, Zhang CL, Gao DS. Glial cell line-derived neurotrophic factor promotes proliferation of neuroglioma cells by up-regulation of cyclins PCNA and Ki-67. Eur Rev Med Pharmacol Sci. 2015;19:2070–75. - PubMed

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