Phosphorylation of histone H3 serine 28 modulates RNA polymerase III-dependent transcription

Abstract

Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell proliferation, transformation and tumor formation. Phosphorylation of histone H3 (H3ph) is induced by tumor promoters (EGF, UV and TPA) and immediate early genes, such as c-myc, c-jun and c-fos. However, it remains to be determined whether H3ph is involved in RNA Pol III transcription. Here, we report that EGF strongly induced H3ph at serine 28 (H3S28ph). EGF significantly increased transcription of RNA Pol III-dependent genes (Pol III genes), tRNA(Leu), tRNA(Tyr), 5S rRNA and 7SL RNA. Inhibition of EGFR, but not PI3K, reduced both H3S28ph and tRNA(Leu) and 5S rRNA transcription. EGF enhanced occupancy of H3S28ph in the promoters of tRNA(Leu) and 5S rRNA. Further analysis indicates that EGF augmented cellular levels of protein and mRNA of TFIIIB subunits, Brf1 and TATA box-binding protein (TBP). Brf1 is a specific transcription factor for RNA Pol III genes. EGF enhanced occupancy of H3S28ph in the Brf1 and TBP promoters. Inhibition of H3S28ph by mutant H3S28A repressed Brf1, TBP and tRNA(Leu) and 5S rRNA expression and decreased occupancy of H3S28ph in their promoters. Reduction of Brf1 significantly decreased tRNA(Leu) and 5S rRNA transcription and repressed EGF-induced anchorage-independent growth. Blocking H3S28ph signaling by using mutant H3S28A reduced EGF-induced cell transformation. Together, these results indicate that EGF activates EGFR signaling to induce H3S28ph, which, in turn, upregulates tRNA(Leu) and 5S rRNA transcription through Brf1 and TBP and promotes cell transformation. The studies demonstrate that epigenetic modification of H3S28ph has a critical role in the activity of Pol III genes.

Fig. 1. EGF-induced H3S28ph requires EGFR, but not PI3K

(A) EGF induces H3S28ph in JB6 cells. JB6 cells were starved in 0.1% FBS/MEM and treated with 0, 5, 10, 20 or 50ng/ml EGF for 30 min. H3S28ph and total H3 were determined by antibodies as designated. (B) EGFR inhibitor reduces H3S28ph. JB6 cells were pretreated with 2μM AG1478 or 25μM LY294002 for 1h and then treated with 20 ng/ml EGF as indicated. H3S28ph.was detected as in A. (C) EGFR deficiency blocks H3S28ph. EGFR-/- and EGFR+/+ MEFs were treated with 20ng/ml EGF and immunoblot analysis was performed using lysates derived from these MEFs as designated. (D) Dominant negative mutant PI3K (ΔPI3K) did not affect H3S28ph. JB6 cells expressing ΔPI3K or vector were treated with EGF to detect H3S28ph as in C. A representative blot from three independent determinations is shown.

Fig. 2. Blocking H3S28ph represses endogenous Pol III gene transcription

(A) EGF enhances Pol III gene transcription in JB6 cells. Cells were treated with 20 ng/ml EGF for 60 min and total RNAs were extracted from these cells. The pre-tRNA^Leu, pre-tRNA^Tyr, 7SL RNA, 5S rRNA, and GAPDH transcripts were measured by RT-qPCR. The fold change was calculated by normalizing to the amount of GAPDH mRNA. (B) Inhibitor of EGFR represses the activity of pre-tRNA^Leu and 5S rRNA genes. The cells were pretreated with AG1478 for 1h and treated with EGF for 60 min. tRNA^Leu and 5S rRNA was determined by RT-qPCR. (C) Inhibition of H3S28ph reduces Pol III gene transcription. JB6 cells were transiently transfected with WT H3 and mutant H3S28A expression constructs for 48h and treated with EGF, the amounts of pre-tRNA^Leu, 5S rRNA, and GAPDH transcripts were measured by RT-qPCR. The bars represent means ± SE of at least three independent determinations.

Fig. 3. EGF enhances expression of TFIIIB subunits

(A) EGF increases cellular levels of TFIIIB subunits in JB6 cells. JB6 cells were treated with EGF. Immunoblot analysis was performed using protein lysates derived from these cells and antibodies against Brf1, TBP, Bdp1, TFIIIC₆₃ and β-actin as designated. A representative blot from three independent determinations is shown. (B) EGF enhances mRNA levels of Brf1, TBP, Bdp1 and TFIIIC₆₃. JB6 cells were treated with EGF. The amounts of Brf1, TBP, Bdp1, TFIIIC₆₃ and GAPDH transcription were measured by RT-qPCR. The values represent means ± SE of three independent determinations.

Fig. 4. EGF induces H3S28ph occupancy in Brf1 and TBP promoters and enhances their expression

(A) EGF enhances occupancy of H3S28ph in the Brf1 promoter. Schematic of the mouse Brf1 promoter and primers used for ChIP assays are designated relative to putative transcription start sites (TSS) and upstream of TSS (top). JB6 cells were treated with EGF and ChIP assays were performed using antibodies to H3S28ph and H3 and qPCR was used to quantify the amplified DNA. The relative occupancy of the proteins was calculated based on the control (no EGF treatment). (B and C) Inhibiting H3S28ph abrogates EGF-enhanced Brf1 and TBP expression. JB6 cells were transfected with WT H3 or mutant H3S28A expression plasmids for 48 hours and then treated with EGF. H3S28ph, H3, c-Myc, Brf1, TBP, TFIIIC₆₃ and β-actin were determined by immunoblot analysis. A representative blot is shown (B). RT-qPCR was performed on RNA isolated from these cells to measure Brf1, TBP, TFIIIC₆₃ and GAPDH transcripts (C). (D) Expression of mutant H3S28A reduces H3S28ph occupancy in Brf1 and TBP promoters. These cells were treated as indicated in B and C. Chromatin was extracted from these cells to perform ChIP assay with H3S28ph antibody. All values shown are the means ± SEM of at least three independent chromatin preparations.

Fig. 5. EGF increases occupancies of Brf1 and H3S28ph, but decreases H3K27me3 in tRNA^Leu and 5S rRNA promoters

(A) EGF-mediated occupancy in the tRNA^Leu promoter. JB6 cells were treated with or without EGF and ChIP assays were performed using Brf1, H3S28ph, H3K27me3 and H3 antibodies and qPCR with specific tRNA^Leu primers to quantify the amplified DNA. (B) EGF enhances Brf1, H3S28ph occupancy in the 5S rRNA promoter. ChIP assays were performed as indicated in Fig. 4 and qPCR was used with 5S rRNA primers to quantify the DNA. (C) Expression of mutant H3S28A reduces H3S28ph occupancy in tRNA^Leu and 5S rRNA promoters. These cells were treated as designed Fig 4B and C. Chromatin was extracted from these cells to perform ChIP assays with H3S28ph antibody. The relative occupancy of the proteins was calculated based on the control (no EGF treatment). All values shown are the means ± SEM of at least three independent chromatin preparations.

Fig. 6. Reduction of Brf1 and H3S28ph represses EGF-induced cell transformation

(A) Repression of Brf1 expression inhibits EGF-induced Pol III gene transcription. JB6 cells were transfected with mismatch (mm) RNA or Brf1 siRNAs for 48 h and treated with EGF. Cell lysates and RNA were isolated from these cells. Immunoblot analysis was performed to determine cellular level of Brf1 protein (left) and pre-tRNA^Leu (middle), 5S rRNA (right) and GAPDH transcripts were measured by RT-qPCR. (B) Down-regulating Brf1 expression decreases EGF-induced anchorage-independent growth. JB6 cells expressing Brf1 siRNAs were poured in triplicate into 6-well plate with 0.35% agar containing 0ng or 20ng/ml EGF. The cells in A were analyzed for growth in soft agar. (C) Blocking H3S28ph signaling represses EGF-induced cell transformation. JB6 stable cell lines expressing pcDNA3 vector (vector), pcDNA3-wild type H3 (WT H3) or pcDNA3-H3S28A (H3S28A) were poured into 6-well plate with EGF as designated at (B). Cells in (A) and (B) were be incubated at 37°C in 5% CO₂ for 1-2 weeks and were fed with fresh complete media with or without EGF twice weekly. Colonies were counted at 1-2 weeks after plating. Values are the means ± SEM (n ≥ 3).

Fig. 7. Schematic illustration of H3S28ph mediating Pol III gene transcription

EGF induces H3S28ph through the EGFR pathway. H3S28ph increases TFIIIB expression, which in turn regulates Pol III gene transcription. H3S28ph also directly upregulates Pol III gene transcription. Both direct and indirect roles of H3S28ph enhance Pol III gene activity to promote cell transformation.