Alcohol induces RNA polymerase III-dependent transcription through c-Jun by co-regulating TATA-binding protein (TBP) and Brf1 expression

Abstract

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

FIGURE 1.

Alcohol induces RNA pol III-dependent transcription. A, ethanol enhances transcription in HepG2-ADH cells. HepG2-vector (HepG2-Vec) and HepG2-ADH cells were serum-starved overnight in 0.1% FBS/DMEM. Cells were treated with or without ethanol, RNA was isolated, and pre-tRNA^Leu, 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, ethanol stimulates transcription in primary mouse hepatocytes. Hepatocytes were treated with or without 50 mm ethanol, RNA was isolated, and the amounts of pre-tRNA^Leu, 5S rRNA, 7SL RNA, and GAPDH transcripts were measured by RT-qPCR. The bars represent means ± S.E. of at least three independent determinations.

FIGURE 2.

Alcohol-mediated activation of JNK1 is required for induction of RNA pol III-dependent transcription. A, ethanol induces JNK1 activation in HepG2-ADH cells. HepG2-vector and HepG2-ADH cells were treated with or without ethanol. Immunoblot analysis was performed using protein lysates derived from these cells and antibodies against phosphorylated JNK1 and -2 (pJNK1 and pJNK2), JNK1, JNK2, and β-actin as designated. A representative blot from three independent determinations is shown. B, ethanol-mediated induction of RNA pol III-dependent transcription requires JNK1 activation. HepG2-ADH cells were pretreated with 5 μm SP600125 and then treated with or without ethanol (left panel). HepG2-ADH cells were transfected with either mismatch or JNK1-specific siRNA for 48 h and then treated with ethanol (right panel). RNA was isolated, and RT-qPCR was performed to measure the amounts of pre-tRNA^Leu, 5S rRNA, and GAPDH transcripts. The -fold change was calculated by normalizing to the amount of GAPDH mRNA. The values represent means ± S.E. of three independent determinations. C, ethanol-mediated JNK1 activation induces expression of TBP and Brf1. Immunoblot analysis was performed using lysates derived from HepG2-ADH cells pretreated with SP600125 (left panel) or transfected with either JNK1 siRNA or mismatch RNA (middle and right panels) as indicated in B. Antibodies against phospho-JNKs, JNKs, TBP, Brf1, TFIIIC₆₃, or β-actin were used as indicated.

FIGURE 3.

Enhanced TBP and Brf1 expression is required for alcohol-mediated induction of RNA pol III-dependent transcription. A, inhibiting ethanol-induced expression of TBP abrogates enhanced RNA pol III-dependent transcription. HepG2-ADH cells were transfected with TBP siRNA (+) or mismatch RNA (−) for 48 h and then treated with ethanol. Resultant protein lysates were subjected to immunoblot analysis using antibodies directed against TBP and β-actin as indicated, and a representative blot is shown (top). RT-qPCR was performed on RNA isolated from these cells to measure pre-tRNA^Leu, 5S rRNA, and GAPDH transcripts (bottom). The -fold change was calculated by normalizing to the amount of transcript in cells transfected with control mismatch RNA. B, ethanol and enhanced TBP expression cooperate to stimulate RNA pol III-dependent transcription. HepG2-ADH cells were transiently transfected with an HA-tagged TBP expression plasmid or control vector for 48 h. Resultant protein lysates were subjected to immunoblot analysis using antibodies directed against TBP and β-actin as indicated (top). RT-qPCR was performed on RNA derived from these cells to measure pre-tRNA^Leu, 5S rRNA, and GAPDH transcripts (bottom). The -fold change was calculated by normalizing to the amount of transcript in cells transfected with vector control plasmid and without ethanol treatment. C, inhibiting ethanol-mediated increases in Brf1 expression abolishes RNA pol III-dependent transcription induction. Experiments were carried out as described in A except that Brf1 siRNA (+) or mismatch RNA (−) was transiently transfected to reduce Brf1 expression, and antibodies against Brf1 were used for the immunoblot analysis shown (top). D, ethanol and enhanced Brf1 expression work together to increase RNA pol III-dependent transcription. Experiments were conducted as described in B, except that an HA-tagged Brf1 expression construct was used to increase Brf1 expression. The values represent means ± S.E. of three independent determinations.

FIGURE 4.

Alcohol-mediated induction of TBP, Brf1, and tRNA gene expression requires increased c-Jun expression. A, ethanol induces an increase in Elk-1 phosphorylation (pElk-1) and c-Jun expression. HepG2-ADH cells were treated with or without ethanol. Immunoblot analysis was performed using protein lysates derived from these cells, and antibodies were used to probe the proteins shown. B, ethanol induces AP-1-dependent promoter activity. HepG2-ADH cells were transfected with a human AP-1-dependent promoter-luciferase construct or the same promoter containing a mutated (Mut) AP-1 site. All cells were cotransfected with a CMV-driven β-galactosidase reporter construct. Resultant cell lysates from cells treated or untreated with ethanol were analyzed for luciferase and β-galactosidase activities. The -fold change in AP-1-dependent promoter activity was calculated relative to untreated control. C, ethanol stimulates Elk-1 transactivation function. HepG2-ADH cells were cotransfected with a Gal4-responsive promoter-luciferase construct containing a vector and either an Elk-1-Gal4 fusion protein expression vector or an empty vector. Cells were treated or untreated with ethanol, and resultant cell lysates were analyzed for luciferase and β-galactosidase activities. -Fold change was calculated based on normalization to luciferase activity in untreated Gal4 reporter cells. All values shown are the means ± S.E. of at least three independent experiments. D, Elk-1 activation is not required for ethanol-mediated induction of TBP, Brf1, and tRNA gene expression. Elk-1-specific siRNAs or mismatch RNAs were transfected into HepG2-ADH cells. Resultant protein lysates derived from cells treated or untreated with ethanol were used for immunoblot analysis to detect Elk-1 and β-actin (top left). RT-qPCR was performed using RNA isolated from these cells to measure Elk-1 mRNA (top right) and tRNA^Leu, TBP, Brf1, and TFIIIC₆₃ transcription (bottom). E, enhanced c-Jun expression is required to drive ethanol-mediated increases in TBP, Brf1, and tRNA gene expression. Top left, same as in D except that HepG2-ADH cells were transfected with c-jun siRNA or mismatch RNA, and immunoblot analysis was used to detect c-Jun and β-actin. RT-qPCR was performed to measure c-Jun mRNA (top right) and tRNA^Leu, TBP, Brf1, and TFIIIC⁶³ transcription (bottom). The values represent means ± S.E. from three independent experiments.

FIGURE 5.

Ethanol induces recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. A, ethanol-mediated binding of transcription components to the TBP promoter. A schematic of the TBP promoter and primers used for ChIP assays is designated relative to the Elk-1 and AP-1 binding sites (top). HepG2-ADH cells were treated with or without ethanol, and ChIP assays were performed using TBP, c-Jun, c-Fos, Elk-1, and H3 antibodies and qPCR to quantify the amplified DNA. The relative occupancy of the proteins was calculated based on the control (no ethanol treatment). All values shown are the means ± S.E. of at least three independent chromatin preparations. B, ethanol enhances c-Jun recruitment to the Brf1 promoter. Top, schematic showing Elk-1 and AP-1 binding sites and the primers used for ChIP assays. Chromatin was isolated from HepG2-ADH cells treated with or without ethanol using antibodies of c-Jun, Elk-1, or H3, and qPCR was used to quantify the DNA. C, ethanol induces c-Jun recruitment to the tRNA^Leu gene. Cells were treated with or without ethanol, and ChIP assays were performed to measure the recruitment of TBP, c-Jun, c-Fos, Elk-1, and H3 on the tRNA^Leu gene promoter. The relative occupancy of the proteins was calculated based on the control (no ethanol treatment). All values shown are the means ± S.E. of at least three independent chromatin preparations.

FIGURE 6.

Chronic alcohol administration in mice induces both TBP-dependent and RNA pol III-dependent transcription. Wild-type C57BL/6 mice or C57BL/6 transgenic mice harboring the NS5A gene that is selectively expressed in hepatocytes were chronically fed with control diet or 3.5% ethanol in the Lieber-DeCarli liquid diet for 12 months. Liver tissues were harvested from these mice, RNA was extracted from non-tumor or tumor portions of the livers, and RT-qPCR was used to measure the amount of pre-tRNA^Leu and 5S rRNA transcripts (top) and TBP and Brf1 transcripts (bottom) relative to GAPDH. The values represent means ± S.E. from three independent experiments. Each group of mice includes at least three mice. The -fold change was calculated in each group by normalizing to wild-type mice fed with control diet.