Role of phosphorylated histone H3 serine 10 in DEN-induced deregulation of Pol III genes and cell proliferation and transformation

Abstract

The products of Pol III genes (RNA polymerase III-dependent genes), such as tRNAs and 5S rRNA, are elevated in both transformed and tumor cells suggesting that they play a crucial role in tumorigenesis. An increase in Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB, augments Pol III gene transcription and is sufficient for cell transformation and tumor formation. We have demonstrated that enhancement of Brf1 and Pol III gene expression is associated with the occurrences of hepatocellular carcinoma (HCC) in mice. This suggests that Brf1 may be a key molecule during HCC development. Diethylnitrosamine (DEN), a chemical carcinogen, has been used to induce HCC in rodents. To determine the role of Brf1 and the epigenetic-regulating events in cell proliferation and transformation, hepatocytes were treated with DEN. The results indicate that DEN increases proliferation and transformation of AML-12 cells. DEN enhanced Brf1 expression and tRNA(Leu) and 5S rRNA transcription, as well as H3S10ph (phosphorylation of histone H3 serine 10). Interestingly, DEN-induced Pol III gene transcription and H3S10ph in tumor cells of liver are significantly higher than in non-tumor cells. Inhibition of H3S10ph by H3S10A attenuates the induction of Brf1 and Pol III genes. Further analysis indicates that H3S10ph occupies the promoters of Brf1 and Pol III genes to modulate their expression. Blocking H3S10ph represses cell proliferation and transformation. These results demonstrate that DEN induces H3S10ph, which mediate Brf1 expression, including but not limited Brf1-dependent genes, to upregulate Pol III gene transcription, resulting in an increase in cell proliferation and transformation.

Fig. 1.

DEN treatment causes the alteration of cell phenotypes. (A) DEN induces colony formation. AML-12 cells were poured in triplicate into six-well plate with 0.35% agar and DEN as indicated. Colonies were counted to analyze cell transformation in soft agar. (B) DEN increases the rate of cell proliferation. AML-12 cells were seeded in into six-well plate and cultured with 100 μg/ml DEN, compared with control cells. The viability and total cell numbers were measured daily for 5 days. *P < 0.05; **P < 0.01.

Fig. 2.

DEN enhances pre-tRNA^Leu and 5S rRNA transcription in mouse hepatocytes. (A) Dose curve of DEN-induced Pol III genes. AML-12 cells were starved in FBS/DMEM-F12 for 4 h. Cells were treated with or without different amounts of DEN for 2 h to extract total RNA and synthesize complementary DNA for RT–qPCR. (B– D) DEN induces Pol III gene transcription AML-12 cells (B), PMHs (C) and tumor stem cells of mouse liver (TSCML) (D) were treated with 100 μg/ml DEN as described. Pre-tRNA^Leu and 5S rRNA, TFIIIC₆₃ (E) and GAPDH transcripts were measured by RT–qPCR. The fold change was calculated by normalizing to the amount of GAPDH mRNA. The bars represent means ± SE of at least three independent determinations. *P < 0.05; **P < 0.01.

Fig. 3.

DEN increases Brf1 expression to modulate Pol III gene transcription. (A) Alteration of Brf1 expression. The total RNAs were extracted from mouse liver tumor tissues or normal liver tissues (n ≥ 4). (B) DEN increases cellular levels of Brf1 in AML-12 cells. AML-12 cells were treated with DEN as described in Figure 1. Total RNA and cell lysates were extracted from the cells. Immunoblot analysis was performed using the protein lysates and antibodies against Brf1 and β-actin as designated. A representative blot from three independent determinations is shown (left). The amounts of Brf1 mRNA were measured by RT–qPCR (right). (C) DEN enhances occupancy of Pol III gene promoters by Brf1. AML-12 cells were treated with DEN to extract chromatins. ChIP assays were performed with Brf1 antibodies, and qPCR was used to quantify the amplified DNA with the specific primers of tRNA^Leu, 5S rRNA and TFIIIC₆₃ (18). (D) siRNA represses Brf1 expression. AML-12 cells were transfected with Brf1 siRNA or mismatch (mm) RNA for 48 h and immunoblot analysis was performed. (E) Repression of Brf1 expression inhibits DEN-induced Pol III gene transcription. AML-12 cells were transfected with mm RNA or Brf1 siRNAs (18) for 48h and treated with DEN. Brf1 mRNA (left), pre-tRNA^Leu (middle), 5S rRNA (right) and GAPDH transcripts were measured by RT–qPCR. The values represent means ± SE of three independent determinations. *P < 0.05.

Fig. 4.

DEM induces H3S10ph and alters the expression of Brf1 and Pol III genes. (A) DEN induces H3S10ph. AML-12, PMHs and TSCMLs were starved in DMEM for 4 h and treated with 100 μg/ml DEN for another 2 h. H3S10ph and total H3 were determined by antibodies as designated. A representative blot from three independent determinations is shown. (B and C) H3S10A expression reduced Brf1 expression and Pol III gene transcription. AML-12 cells were transfected with WT H3 or mutant H3S10A expression constructs for 48 h. The cells were treated with 100 μg/ml DEN. Total RNAs and cell lysates were extracted from these cells. Immunoblot analysis was performed to determine H3S10ph, H3, Brf1, TFIIIC63 and β-actin with the antibodies as designed. A representative blot is shown (B, left). Brf1 mRNA (B, right), pre-tRNA^Leu (C, left) and 5S rRNA (C, right) and GAPDH transcripts were measured by RT–qPCR. All values shown are the means ± SEM of at least three independent experiments.

Fig. 5.

DEN increases occupancies of H3S10ph to the promoters of Brf1 and Pol III genes. (A) Schematic of the mouse Brf1 promoter. The primers used for ChIP assays are designed relative putative transcription start sites in the Brf1 promoter of mouse. (B) DEN treatment increases H3S10ph to Brf1 at −71 bp, not −1630 bp. (C) Expression of mutant H3S10A reduces H3S10ph occupancy in tRNA^Leu and 5S rRNA promoters. AML-12 cells were transfected with WT H3 or mutant H3S10A expression constructs for 48 h, and the cells were treated with or without DEN for another 2 h. ChIP assays were performed using H3S10ph and H3 antibodies and qPCR with specific primers (18) recognizing different regions of Brf1, tRNA^Leu and 5S rRNA promoters. All values shown are the means ± SE of at least three independent chromatin preparations.

Fig. 6.

Expression of mutant H3S10A reduces DEN-induced transformation. (A) Reduction of cell transformation. Stable AML-12 cells expressing pcDNA3 vector (vector), pcDNA3-WT H3 (WT H3) or pcDNA3-H3S10A (H3S10A) were poured in triplicate into six-well plate with 0.35% agar containing 100 μg/ml DEN and control. The cells were analyzed for colony formation in soft agar. **P < 0.01. (B) Blocking H3S10ph signaling represses DEN-induced cell proliferation. AML-12 stable cell lines expressing pcDNA3 vector (vector), pcDNA3-WT H3 or pcDNA3-H3S10A were poured into six-well plate with 100 μg/ml DEN. The viability and total cell numbers were measured daily for 5 days. Values are the means ± SE of three independent experiments.

Fig. 7.

Schematic illustration of H3S10ph mediating Pol III gene transcription. DEN induces H3S10ph to increase Brf1 expression, which in turn regulates Pol III gene transcription indirectly. H3S10ph also directly upregulates Pol III gene transcription. Both direct and indirect roles of H3S10ph enhance Pol III gene activity to promote cell proliferation and transformation.