The MTE, a new core promoter element for transcription by RNA polymerase II

Abstract

The core promoter is the ultimate target of the vast network of regulatory factors that contribute to the initiation of transcription by RNA polymerase II. Here we describe the MTE (motif ten element), a new core promoter element that appears to be conserved from Drosophila to humans. The MTE promotes transcription by RNA polymerase II when it is located precisely at positions +18 to +27 relative to A(+1) in the initiator (Inr) element. MTE sequences from +18 to +22 relative to A(+1) are important for basal transcription, and a region from +18 to +27 is sufficient to confer MTE activity to heterologous core promoters. The MTE requires the Inr, but functions independently of the TATA-box and DPE. Notably, the loss of transcriptional activity upon mutation of a TATA-box or DPE can be compensated by the addition of an MTE. In addition, the MTE exhibits strong synergism with the TATA-box as well as the DPE. These findings indicate that the MTE is a novel downstream core promoter element that is important for transcription by RNA polymerase II.

Figure 1.

Identification of core promoters that contain a motif 10 sequence. (A) Putative core promoters that contain a motif 10 consensus sequence were identified in the Drosophila genome database by using the JDSA search program. In these putative core promoters, the motif 10 consensus (in red) is located from +18 to +29 relative to the A₊₁ position in the initiator (Inr) consensus sequence (in blue). (B) Identification of five motif 10-containing core promoters. The in vivo start sites were mapped by primer extension analysis with poly(A)⁺ RNA (15 μg) from Drosophila embryos that were collected from 0 to 12 h after egg deposition. The in vitro start sites were mapped with RNA that was synthesized in vitro with a nuclear extract derived from Drosophila embryos. Where indicated, α-amanitin (α-am; 4 μg/mL) was included in the in vitro transcription reactions. For each promoter, the primer extension products were subjected to denaturing polyacrylamide gel electrophoresis in parallel with DNA sequencing ladders that were generated from the same primers that were used in the primer extension analyses of the RNA. Transcription from each of these promoters appears to start from the C nucleotide that is immediately upstream of the “A₊₁” position of the Inr consensus. For consistency of nomenclature, however, we will continue to refer to “A₊₁” as the “+1” position of these promoters.

Figure 2.

Scanning mutational analysis of the motif 10 sequence. A series of mutant CG4427 core promoters was constructed in which triple nucleotide substitutions were introduced in the downstream promoter region that encompasses the motif 10 sequence and the DPE. Outside the motif 10 sequence, A, T, and G nucleotides were mutated to C, and C nucleotides were mutated to A. Within the motif 10 sequence, the substitution mutations were designed to minimize the similarity of the sequence to the motif 10 consensus. The wild-type and mutant promoters were subjected to in vitro transcription analysis with a Drosophila nuclear extract. The transcriptional activity of each mutant promoter is reported relative to that of the wild-type promoter.

Figure 3.

Nonoverlapping mutations in the motif 10 sequence and the DPE. (A) The +30–33 DPE mutation inactivates DPE-dependent promoters. The +30 to +33 region of the E74B and Doc core promoters was mutated to CATA. The resulting m30–33 promoters were subjected to in vitro transcription analysis in parallel with the corresponding wild-type promoters. (B) Analysis of mutations in the motif 10 sequence (+19 to +29) that do not overlap with the DPE (+28 to +33). A series of progressive substitution mutant versions of the Tollo core promoter was constructed, as depicted at the bottom of the figure. The substitution mutations were chosen to minimize the similarity of the sequences to the motif 10 consensus. The wild-type and mutant promoters were subjected to in vitro transcription analysis with a Drosophila nuclear extract.

Figure 4.

The motif ten element, MTE, supports transcription in the absence of the DPE. (A) Diagram of the MTE (m18–22) and DPE (m30–33) mutations. The MTE is depicted from +18 to +27, because this segment of the motif 10 consensus is sufficient to confer MTE activity (see below). (B) The MTE and DPE motifs both contribute to transcription from the Tollo, CG10479, and CG15695 core promoters. The wild-type, m18–22, m30–33, and m18–22/30–33 versions of each core promoter were subjected to in vitro transcription analysis with a Drosophila nuclear extract. (C) The E74B and Doc core promoters lack a functional MTE that can support transcription upon mutation of the DPE. Wild-type and mutant promoters were analyzed, as in B.

Figure 5.

The MTE functions with the Inr in a spacing-dependent manner. (A) The Inr is required for transcription from MTE-containing promoters. In the mutant Inr (mInr) promoters, the Inr sequences (shown in Fig. 1) were mutated to GTGACA. The constructs were subjected to in vitro transcription analysis with a Drosophila nuclear extract. (B) The spacing between the Inr and the MTE is important for core promoter activity. A series of mutant promoters was constructed in which the spacing between the Inr and the MTE was either increased or decreased by one or three nucleotides, as depicted. To ensure that the effects are due to interactions between the Inr and MTE, all of the promoters contain the m30–33 mutation (CATA at +30–33) that inactivates the DPE motif. The constructs were subjected to in vitro transcription analysis with a Drosophila nuclear extract. The transcriptional activity of each mutant promoter is reported relative to that of the m30–33 promoter with wild-type spacing (0) between the Inr and the MTE.

Figure 6.

The MTE can compensate for the loss of a DPE. The diagram depicts the four variants of the E74B and Doc core promoters that were tested. In the constructs that contain an MTE (Inr–MTE and Inr–MTE–DPE), the +18 to +27 segment of each wild-type promoter is replaced by the MTE sequence (from +18 to +27) of the Tollo core promoter. In the Inr and Inr–MTE constructs, the DPE sequence is mutated to CATA at positions +30 to +33. These E74B and Doc promoter sets were subjected to in vitro transcription analysis with a Drosophila nuclear extract.

Figure 7.

The MTE can compensate for the loss of a TATA-box. Two sets of hybrid promoters were constructed by fusing the -36 to +10 region of the hbP2 promoter to the +16 to +40 region of either the Tollo or the CG10479 promoter. The hbP2 core promoter contains repeated Inr motifs that direct initiation at +1 and +5. In the hybrid promoters, the spacing of the MTE motifs is aligned with the Inr at +1. To eliminate the contribution of the DPE, all of the promoters contain the m30–33 mutation (CATA at +30 to +33) that inactivates the DPE. The Inr and Inr–MTE constructs contain the mTATA mutation, in which the hbP2 TATA-box, TATATAAA, is replaced by ACGTCCGT. The TATA–Inr and Inr constructs contain the m18–22 mutation (ATCCA from +18 to +22), which inactivates the MTE. The hybrid promoter sets were subjected to in vitro transcription analysis with a Drosophila nuclear extract, and the transcriptional activity of each hybrid promoter is reported relative to that of the TATA–Inr (“wild-type” hbP2) promoter.

Figure 8.

The MTE appears to affect the interaction of TFIID with the core promoter. The wild-type, m18–22 (mutant MTE), and m18–22/30–33 (mutant MTE and DPE) versions of the Tollo core promoter were subjected to DNase I footprinting analysis with purified Drosophila TFIID. The mutation of the MTE in the absence of the DPE results in the decrease of a TFIID-induced hypersensitive site (arrow) as well as the loss of sites of weak protection by TFIID (dots) in the vicinity of the Inr (cf. lanes 3,4 and 5,6).

Figure 9.

Human transcription factors recognize the MTE. (A) The MTE in the Drosophila Tollo core promoter is recognized by human basal transcription factors. The Tollo promoter constructs described in Figure 4 were transcribed with a HeLa nuclear extract. (B) Transient transfection analysis of wild-type and mutant Tollo core promoters in HeLa cells. The wild-type, m18–22, m30–33, and m18–22/30–33 versions of the Tollo–luc reporter constructs as well as the promoterless vector (pGL3-Basic; “vector only”) were transiently transfected in HeLa cells, and the relative activities were determined. (C) The MTE exhibits synergy with the TATA-box as well as weak activity in the absence of the TATA and DPE motifs with human transcription factors. The hbP2–Tollo hybrid promoter constructs shown in Figure 7 were transcribed with a HeLa nuclear extract. (D) The MTE can compensate for the loss of the DPE and exhibits synergy with the DPE with human transcription factors. The E74B-based constructs shown in Figure 6 were transcribed with a HeLa nuclear extract. (E) The human sterol C5 desaturase-like (SC5DL) gene has an MTE-dependent promoter. The wild-type, m18–22, m30–33, and m18–22/30–33 versions of the SC5DL promoter were subjected to in vitro transcription analysis with a HeLa nuclear extract.