Epigenetic modifications promote the expression of the orphan nuclear receptor NR0B1 in human lung adenocarcinoma cells

Abstract

The ectopic activation of NR0B1 is involved in the development of some cancers. However, the regulatory mechanisms controlling NR0B1 expression are not well understood. Therefore, the epigenetic modifications promoting NR0B1 activation were examined in this study. NR0B1 protein was detected in cancerous tissues of more than 50% of human lung adenocarcinoma (ADCA) cases and tended to be expressed in low-differentiated cancerous tissues obtained from males. Nevertheless, NR0B1 activation in ADCA has not previously been correlated with DNA demethylation. NR0B1 expression was not detected in 293T cells, although it contains a hypomethylated NR0B1 promoter. Treating 293T cells with a histone deacetylase inhibitor increased acetylated histone H4 binding to the NR0B1 promoter and activated NR0B1 expression. In contrast, treatment with histone methylase inhibitors decreased the methylation of histones H3K9 and H3K27 and slightly induced NR0B1 transcription. Furthermore, the level of acetyl-histone H4 binding to the NR0B1 promoter increased, whereas the occupancy of H3K27me3 was lower in cancerous tissues than in non-cancerous tissues. Similar histone occupancies were confirmed in a comparison of cancerous tissues with strong, moderate and negative NR0B1 expression. In conclusion, this study shows that CpG methylation within the NR0B1 promoter is not involved in the in vivo regulation of NR0B1 expression, whereas the hyperacetylation of histone H4 and the unmethylation of histones H3K9 and H3K27, and their binding to the NR0B1 promoter results in decondensed euchromatin for NR0B1 activation.

Keywords: DNA methylation; NR0B1; cell self-renewal; histone modifications; lung adenocarcinoma.

Figure 1. Expression profile of the NR0B1 protein in human lung adenocarcinoma samples

A. An example showing strong NR0B1-positive staining in cancerous tissue from a male case of clinical stage III cancer but not in the adjacent non-cancerous tissue B, C. An example showing strong NR0B1-positive staining in cancerous tissue from a female case of clinical stage III cancer but not in the adjacent non-cancerous tissue D, E-F. An example showing a moderate immunoreactive signal for NR0B1 in the cancerous tissues from one male case (E) and one female case (F) of clinical stage II cancer. G-H. Representative image showing NR0B1-negative staining in cancerous tissues (from one female case in stage III (G) and one male case in stage I cancer (H)). Scale bar = 10 μm. I. Varying amounts of NR0B1 protein were detected in different male cancerous tissues that were obtained from several representative cases of stages I, II and III cancer and analyzed using immunoblotting. J. Relative NR0B1 protein levels (versus GAPDH) in male cancerous tissues in stage II and stage III (14 cases/stage). The results showed that the NR0B1 protein was present at a higher level in the male stage III cancerous tissues than the male stage II cancerous tissues.

Figure 2. Methylation status of CpG sites within the NR0B1 promoter in human samples

A. Scheme showing the CpG sites within the NR0B1 promoter. The numbers indicate the positions of the CpG sites, with the transcription start site (TSS) considering to be +1. B. The pattern of methylation of the NR0B1 CpGs in cancerous (−c) and normal non-cancerous (−p) tissues in male and female lung adenocarcinoma cases. Cases 1 and 3: Clinical stage III with strong NR0B1 expression; and cases 2 and 4: stage II with moderate NR0B1 expression. C. Cases (negative) showing a mixture of cancerous tissues with a negative NR0B1 signal from 10 male (−m) and female (−f) cases, respectively. D. The NR0B1 methylation status is shown for the genomic DNA from male (−m) and female (−f) peripheral blood and testis samples. Empty squares indicate unmethylated cytosine residues, and shaded squares indicate methylated cytosine residues. A.M.: average ratio of methylated CpG sites.

Figure 3. Correlation analysis of NR0B1 expression with its CGI methylation status in nine cell lines

A. The NR0B1 mRNA was detected using RT-PCR in A549, DU145 and SKOV-3 cells but not in 293T, HepG2, LNCaP, PC-3, MCF7 and HeLa cells. NC: Water acted as a negative control for the RT-PCR template. B. Relative methylated levels of the NR0B1 CGIs in different cells. M: DNA ladder. A.M.: average ratio of methylated CpG sites.

Figure 4. DNA demethylation activates NR0B1 expression

A. The expression of NR0B1 was activated in PC-3 and MCF7 cells that were treated with the DNA methyltransferase inhibitor AZA. NC: Water acted as a negative control for the RT-PCR template. B. NR0B1 CGI was partially demethylated in cells that were treated with AZA. A.M.: average ratio of methylated CpG sites.

Figure 5. Histone H4 acetylation promotes NR0B1 gene expression

A. The expression of NR0B1 mRNA was activated in 293T cells that were treated with the HDAC inhibitor TSA (upper panel: gel electrophoresis results of RT-PCR; low panel: qRT-PCR results). B. Protein levels of acetyl-histone H3 and H4 and the transcription factors AR, NANOG, NR5A1, NR5A2, OCT3/4 and SOX2 were compared before and after TSA treatment in 293T cells. TUBULIN acted as an internal control. C. Relative protein amounts (versus TUBULIN) of NR0B1, acetyl-histone H3 and H4, AR, NANOG, NR5A1, NR5A2, OCT3/4 and SOX2 in 293T cells before and after TSA treatment. D. ChIP-qPCR analysis of the NR0B1 promoter region using anti-acetyl-histone H3 and H4 antibodies in 293T cells before and after TSA treatment. The promoter region from −267 to −156 of the NR0B1 gene was significantly enriched in TSA-treated 293T cells, as detected using anti-acetyl-histone H4 antibodies. * p value <0.05 and ** p value <0.01 in the Student's t-test.

Figure 6. Synergistic effects of AZA and TSA on NR0B1 gene activation

A-B. TSA further increased NR0B1 expression in PC-3 and MCF7 cells to a higher level than treatment with AZA alone. Treatment with TSA alone did not activate NR0B1 expression. C. The DNA methylation level of the NR0B1 CGI in PC-3 cells that were treated with AZA, TSA alone, or a combination of AZA and TSA. A.M.: average ratio of methylated CpG sites.

Figure 7. Effect of histone demethylation on NR0B1 expression

A-B. Treatment with the histone methylase inhibitors DZNeP (DZNP) and BIX01294 (BIX) resulted in slightly higher levels of NR0B1 expression in 293T cells than was induced by treatment with the vehicle (DMSO). A549 acted as a positive control and water was used as a negative control (NC) in the RT-PCR analysis. C. The protein levels of the DZNep target methylase EZH2, the BIX01294 target methylase EHMT2, and H3K9me2 and H3K27me3 were detected in 293T cells that were treated with DZNeP, BIX01294 or vehicle (DMSO). * p value <0.05, ** p value <0.01 and *** p value <0.001 using the Student's t-test.

Figure 8. Histone modifications within the NR0B1 promoter in different tissues and cells by ChIP-qPCR analysis

Different histone modifications were detected in the NR0B1 promoter region between 293T and A549 cells A. the side (SP) and main (MP) populations of A549 cells B. cancerous and non-cancerous tissues C. cancerous tissues with a strong and negative NR0B1 signal D. cancerous tissues with a strong and moderate NR0B1 signal E. and between testis tissues and peripheral blood F. The cancerous and non-cancerous tissues were mixed samples from 5 patients. * p<0.05, ** p value <0.01 and *** p value <0.001 using the Student's t-test.

Figure 9. Proposed model for the in vivo epigenetic regulation of NR0B1 expression

A-B. Hypoacetylation of histone H4 and H3K9me2 (A) / H3K27me3 (B) binding to the NR0B1 promoter silences NR0B1 transcription, although the NR0B1 CGI is unmethylated. C. In addition to unmethylated the NR0B1 CGIs, hyperacetylated histone H4 and the demethylated lysines 9 and 27 of histone H3 on the NR0B1 promoter facilitate its activation to promote cell self-renewal.