TRF2, but not TBP, mediates the transcription of ribosomal protein genes - PubMed (original) (raw)

TRF2, but not TBP, mediates the transcription of ribosomal protein genes

Yuan-Liang Wang et al. Genes Dev. 2014.

Abstract

The TCT core promoter element is present in most ribosomal protein (RP) genes in Drosophila and humans. Here we show that TBP (TATA box-binding protein)-related factor TRF2, but not TBP, is required for transcription of the TCT-dependent RP genes. In cells, TCT-dependent transcription, but not TATA-dependent transcription, increases or decreases upon overexpression or depletion of TRF2. In vitro, purified TRF2 activates TCT but not TATA promoters. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) experiments revealed the preferential localization of TRF2 at TCT versus TATA promoters. Hence, a specialized TRF2-based RNA polymerase II system functions in the synthesis of RPs and complements the RNA polymerase I and III systems.

Keywords: RNA polymerase II; TCT motif; TRF2; core promoter; ribosomal protein genes.

© 2014 Wang et al.; Published by Cold Spring Harbor Laboratory Press.

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Figures

Figure 1.

Figure 1.

TCT-dependent transcription appears to require TRF2 but not TBP. (A) Schematic diagrams of hTRF2 and the two forms of Drosophila TRF2 (dTRF2S and dTRF2L). dTRF2S is identical to the C-terminal 632-amino-acid residues of dTRF2L. (B) Depletion of TRF2, but not TBP, reduces RP gene expression. Drosophila S2 cells were depleted of either TRF2 or TBP by RNAi and then transfected with TCT-dependent or TATA-dependent luciferase reporter genes. The experimental scheme and reporter constructs are depicted at the bottom of the figure. The activities of the RNAi-depleted extracts are reported as relative to the activities of mock RNAi-treated control extracts. Error bars represent the standard deviation. (C) Analysis of endogenous transcript levels by qRT–PCR. Drosophila S2 cells were depleted of TRF2 or TBP, as in B. The total RNA was then isolated and analyzed by qRT–PCR. For the RP genes, intronic sequences were used to detect newly synthesized transcripts. We did not analyze RP genes with intronic snoRNA genes, as they could affect the levels of the intronic RNAs. The error bars represent the standard deviation.

Figure 2.

Figure 2.

Purified TRF2 is required for in vitro transcription of TCT-dependent genes but not a TATA-dependent gene. (A) Immunodepletion of endogenous dTRF2 from a Drosophila embryo nuclear extract. The levels of TRF2 (dTRF2S), TBP, TFIIB, and TFIIA (p30 subunit) in TRF2-depleted extracts versus control extracts were monitored by Western blot analysis. We were able to detect dTRF2S but not dTRF2L even though the antibodies were raised against a polypeptide that is shared by both proteins. This effect may be due to inefficient transfer of the dTRF2L protein to the blot, the lack of recognition of dTRF2L by the antibodies, or the absence of dTRF2L in the extract used in the Western blot. (B) Purified hTRF2, but not purified hTBP, is able to restore the specific loss of TCT-dependent transcription that occurs upon depletion of TRF2 from a nuclear extract. Two-template in vitro transcription assays were performed with TCT-dependent and TATA-dependent promoters. Reactions were carried out with either TRF2-depleted or control nuclear extracts. Where indicated, purified hTRF2 or hTBP was added to reactions with the TRF2-depleted extracts. The resulting transcripts were detected by primer extension–reverse transcription analysis. The single asterisk denotes a nonspecific transcript. The double asterisk indicates a nonspecific transcript that is observed in the presence of hTBP. This stimulation of nonspecific initiation by hTBP is most likely a consequence of the relatively nonspecific binding of hTBP to DNA. This effect was not observed with dTBP (Supplemental Fig. S7).

Figure 3.

Figure 3.

Overexpression of TRF2 increases TCT-dependent but not TATA-dependent transcription, whereas overexpression of TBP increases TATA-dependent but not TCT-dependent transcription. Drosophila S2 cells were transfected with a TCT-dependent or TATA-dependent reporter construct along with the indicated amounts of expression vector for either dTRF2S or dTBP. The AdML-_Antp_P2 promoter is identical to the TATA promoter that was used in Figure 1B. Luciferase reporter activities were normalized to those obtained with the empty vector alone. Error bars represent the standard deviation.

Figure 4.

Figure 4.

TRF2 is enriched at TCT-dependent promoters in vivo. The occupancy of TRF2 and TBP was analyzed by ChIP-seq experiments with Drosophila embryos collected from 2 to 4 h after egg deposition. (A) Differential TRF2 occupancy at a TATA promoter and a TCT promoter. Read counts across a representative TATA-containing promoter (achaete) and a representative TCT-containing promoter (RpL30) with comparable levels of RNA Pol II show that TRF2 is bound at higher levels to the TCT promoter relative to the TATA promoter. (B) Heat maps of the ChIP occupancy of Pol II, TRF2, and TBP at 171 genes with a predicted TATA box and at 134 genes with a predicted TCT motif. These two sets of genes were sorted in descending order of Pol II occupancy and are shown in a window from −200 to + 800 nt relative to the +1 transcription start site. The same genes and their order are shown for Pol II, TBP, and TRF2 occupancy. (C) TRF2 occupancy is highest near the transcription start site of RP genes. The graph depicts the average enrichments of the TRF2 and TBP ChIP-seq signals over input from −250 to +500 nt relative to the transcription start site for the 87 known RP genes. The dashed line indicates the peak TRF2 signal at −3 nt relative to the transcription start site.

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