Characterization of sINR, a strict version of the Initiator core promoter element

Abstract

The proximal promoter consists of binding sites for transcription regulators and a core promoter. We identified an overrepresented motif in the proximal promoter of human genes with an Initiator (INR) positional bias. The core of the motif fits the INR consensus but its sequence is more strict and flanked by additional conserved sequences. This strict INR (sINR) is enriched in TATA-less genes that belong to specific functional categories. Analysis of the sINR-containing DHX9 and ATP5F1 genes showed that the entire sINR sequence, including the strict core and the conserved flanking sequences, is important for transcription. A conventional INR sequence could not substitute for DHX9 sINR whereas, sINR could replace a conventional INR. The minimal region required to create the major TSS of the DHX9 promoter includes the sINR and an upstream Sp1 site. In a heterologous context, sINR substituted for the TATA box when positioned downstream to several Sp1 sites. Consistent with that the majority of sINR promoters contain at least one Sp1 site. Thus, sINR is a TATA-less-specific INR that functions in cooperation with Sp1. These findings support the idea that the INR is a family of related core promoter motifs.

Figure 1.

(A) sINR is located mainly around the TSS. The distribution of sINR at 5 nt intervals throughout the proximal promoter region (−60 to +40 relative to the TSS) as determined by the DBTSS. (B) The sINR is a strict consensus and differs from other known INRs in its flanking sequences. The upper panel shows a graphical representation of multiple sequence alignment (http://weblogo.berkeley.edu/logo.cgi) of sINR from 112 genes out of 191 that contain the element around their TSSs. Alignment of all 191 genes resulted in the same consensus (data not shown). Comparison of the sINR consensus to the broad INR consensus (middle panel) and to INR sequences of subset of genes that contain functional INR in their promoter (lower panel).

Figure 2.

sINR is essential for transcription directed by DHX9 and ATP5F1 promoters. (A) Determination, by primer extension, of the TSSs of the endogenous DHX9 and ATP5F1 genes using gene-specific primers as probes and total RNA prepared from 293T cells. The primer-extension products were run together with sequencing ladders (marked A, C, G and T). The TSSs are indicated by arrowheads and their positions are shown in (B). (B) The DNA sequences and the positions of TSSs of the DHX9 and ATP5F1 promoters. The TSSs are indicated by arrows and correspond to the TSS bands shown in (A). The sINR motif is underlined and the lower case letters underneath indicate the sequence of the mutation as shown in (C). (C) The effect of sINR mutation on transcription. The promoters of the DHX9 and ATP5F1 genes (from −150 to +50 and −55 to +60 of DHX9 and ATP5F1, respectively) were cloned in front of a firefly luciferase reporter gene and then subjected to site-directed mutagenesis to create sINR mutants. The wild type (WT) or mutated (mut) promoter or the promoter-less parental plasmid (pGL2-basic, C) was co-transfected into 293T cells with RSV-renilla luciferase that serves as a reference for transfection efficiency. Twenty-four hours post transfection firefly and renilla luciferase activities were measured. The results shown are the average ± SD of four independent experiments, the activity of the wild type promoters being 100%. Primer extension analysis of the wild type or mutated DHX9 promoter is shown in Supplementary Data 2.

Figure 3.

The sequence requirements for sINR function. (A) A scheme of sINR mutants. The mutated sequences within sINR of the DHX9 promoter are shown in lower case letters. (B) The wild type and mutated promoters (fused to firefly luciferase reporter gene) were transfected into 293T cells together with RSV-renilla that serves as internal control. Twenty-four hours post transfection firefly and renilla luciferase activities were measured. The normalized results are the mean of at least four independent experiments (±SD), wild type DHX9 promoter activity being 100%. (C) Position of the TSS of wild type (WT) and the indicated representative mutants were determined by primer extension. The primer-extension products were run together with sequencing ladders (A, C, G and T).

Figure 4.

Functional relationship between sINR and INR. (A) The core or the full sINR of DHX9 promoter was mutated to fit the AdML INR sequence as shown in the upper panel. The wild type and mutated promoters were analyzed as in Figure 3. The results are average of four independent experiments (±SD). (B) The INR of the Pel98 promoter was either mutated or replaced by sINR as shown in the upper panel. The Pel98 promoter derivatives (fused to firefly luciferase reporter gene) were transfected into MEFs together with RSV-renilla that serves as internal control. Twenty-four hours post transfection firefly and renilla luciferase activities were measured. The results are average of four independent experiments (±SD).

Figure 5.

(A) The sequence of DHX9 wild type and linker mutated constructs. (B) Firefly luciferase reporter gene driven by the DHX9 promoter and the linker mutant derivatives and the promoter-less reporter were transfected into 293T cells together with RSV-renilla luciferase that served as control for transfection efficiency. Twenty-four hours later firefly and renilla luciferase activities were measured and the relative activity is presented as ratio of DHX9 wild type promoter. Control indicates the activity of the promoter-less reporter, ‘ds’ and ‘us’ denote downstream and upstream, respectively. (C) The effect of DHX9 promoter linker mutants on TSS selection. Wild type and mutated constructs were transfected into 293T cells together with puro-GFP and their mRNA levels were monitored by primer extension and normalized with puro-GFP mRNA. Control indicates the activity of the promoter-less reporter, ‘ds’ and ‘us’ denote downstream and upstream, respectively. The positions of the TSSs are shown in (A) by arrows.

Figure 6.

Defining the minimal sequence for DHX9 promoter with activity. (A and B) Schematic representation of promoter dissections (right panel). The positions of Sp1 and sINR are indicated. The reporter constructs were transfected into 293T cells and promoter activity was measured by luciferase assay (A) and primer extension (B) (left panel). (C) The inverted repeat sequence located at −49 to −37 of the DHX9 promoter (each repeat is indicated by an overhead line) and its mutant derivative. The promoter activity of these constructs after transfection to 293T cells was measured by the luciferase assay (lower panel). (D) The importance of the distance between sINR and the inverted repeat element. A 5-bp linker was inserted between sINR and the inverted repeat sequence (upper panel). Promoter activity, after transfection into 293T cells of the wt or the linker-bearing constructs, was measured by the luciferase assay. (E) The frequency of TATA, DPE and Sp1 in sINR-containing genes. These elements were searched for in sINR promoters using the minimal TATA box consensus TATAAA, the DPE consensus RGWYV and Sp1 concensus CCCCGCCCC allowing up to two mismatches, at their defined locations: TATA −35 to −25; DPE at +28, in addition to a G residue at position +14; Sp1 at position −300 to +100 in both strands relative to the TSS. The frequency of the motif in sINR promoters is presented as a percentage.

Figure 7.

sINR can substitute for the TATA-like element of the early SV40 promoter. (A) The TATA-like core promoter of the early SV40 promoter was replaced with either a random sequence (control), sINR sequence or canonical TATA box (upper panel). The SV40 promoter derivatives were transfected into 293T cells together with CMV puro-GFP and analyzed by primer extension 24 h later (lower panel). (B) The effect of mutations in sINR in the heterologous context of the SV40 promoter. Mutated sINR sequences (top panel) were analyzed by the luciferase assay. Normalized results (mean ± SD) of four independent experiments, the activity of the wild type sINR being 100%, are shown in the lower panel.

Figure 8.

Transcription factor YY1 binds sINR but is dispensable for sINR function. (A) EMSA using HeLa cell nuclear extract and a fluorescently labeled double stranded oligonucleotide containing DHX9 sINR. Lane 1 is the probe and lane 2 is the probe incubated with the nuclear extract. Competitor DNAs were added to the reactions in lanes 3–8 as indicated on the top. The specific protein–DNA complex and the free probes are indicated by arrows. The sequences of oligos used for binding and competition are shown in the left panel. (B) EMSA as in (A) with anti-YY1 and anti-TBP antibodies added to the reactions as indicated. (C) HeLa cells were transfected with YY1, YY2 or both (YY1 + YY2) siRNA expression plasmids or with the parental plasmid (control) along with 100 ng of puro-GFP. Twenty-four hours later puromycin was added for selection and after an additional 48 h the cells were harvested and subjected to RNA and protein extract preparation. YY1 depletion was monitored by western blot using YY1 and tubulin antibodies (left panel). YY2 depletion was monitored by real time PCR analysis using specific primers for the YY2 gene (right panel) and normalized to GAPDH mRNA. (D) DHX9, c-myc and GAPDH mRNA levels were quantified by real time PCR analysis using specific primers. DHX9 and c-myc levels were normalized to GAPDH and the mean ± SD of their relative levels from four independent transfection experiments are presented. (E) 293T cells were transfected with YY1 siRNA expression plasmid (siYY1) or with parental plasmid (control). Forty-eight hours later the cells were transfected again with either the YY1 siRNA expression plasmid or the control together with DHX9-luciferase reporter plasmid. Twenty hours later the cells were harvested and subjected to RNA and protein extract preparations. Luciferase mRNA level was quantified by real time PCR and the graph shows the mean ± SD of three independent transfection experiments. Normalization of transfection efficiency was done by measuring the mRNA level of the neomycin resistant gene under the SV40 early promoter that was co-transfected to the cells.

Figure 9.

Dependency of the DHX9 promoter on TAF1. Hamster ts13 temperature sensitive cells were co-transfected with the indicated reporter plasmids. The cells were incubated at the permissive temperature (32°C) for 6 h, washed and then separated into two groups. One was grown at 32°C and the second at the nonpermissive temperature (39.5°C) for an additional 48 h, after which the cells were harvested and luciferase activity was measured. RSV promoter-driven renilla reporter luciferase plasmids served to normalize transfection efficiency. Results are the mean of three independent experiments, each with independent duplicates. The asterisk denotes that the difference of the DHX9 promoter activity at the different temperatures is significant, P < 0.01.