doi: 10.1101/gad.1698108.
Caudal, a key developmental regulator, is a DPE-specific transcriptional factor
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- PMID:18923080
- PMCID: PMC2569877
- DOI: 10.1101/gad.1698108
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Caudal, a key developmental regulator, is a DPE-specific transcriptional factor
Tamar Juven-Gershon et al. Genes Dev..
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Abstract
The regulation of gene transcription is critical for the proper development and growth of an organism. The transcription of protein-coding genes initiates at the RNA polymerase II core promoter, which is a diverse module that can be controlled by many different elements such as the TATA box and downstream core promoter element (DPE). To understand the basis for core promoter diversity, we explored potential biological functions of the DPE. We found that nearly all of the Drosophila homeotic (Hox) gene promoters, which lack TATA-box elements, contain functionally important DPE motifs that are conserved from Drosophila melanogaster to Drosophila virilis. We then discovered that Caudal, a sequence-specific transcription factor and key regulator of the Hox gene network, activates transcription with a distinct preference for the DPE relative to the TATA box. The specificity of Caudal activation for the DPE is particularly striking when a BRE(u) core promoter motif is associated with the TATA box. These findings show that Caudal is a DPE-specific activator and exemplify how core promoter diversity can be used to establish complex regulatory networks.
Figures
The core promoters ofDrosophila Hox genes contain a conserved DPE motif. The core promoter sequences of the indicatedD. melanogaster Hox genes are shown. We initially analyzed the sequence conservation of the promoters in eight differentDrosophila species by using VISTA tools and CLUSTALW (Supplemental Fig. 1; data not shown). Based on this sequence alignment, positions that are identical betweenD. melanogaster andD. virilis (in terms of sequence and spacing relative to the A + 1 in the Inr) are shaded.D. melanogaster andD. virilis are estimated to be separated by an evolutionary period of ∼40–60 million years.
The DPE is present in many genes that regulate the development of theDrosophila body plan. (A) Most of the Hox genes contain DPE-dependent core promoters. A schematic diagram of the Hox gene cluster is shown at thetop. (lab)labial; (pb)proboscipedia; (Dfd)Deformed; (Scr)Sex combs reduced; (Antp P1)Antennapedia upstream promoter; (Antp P2)Antennapedia downstream promoter; (Ubx)Ultrabithorax; (abd-A)abdominal-A; (Abd-B)Abdominal-B. The wild-type (wt) and mutant DPE (mDPE) versions of the indicated core promoters (from −10 to +40 relative to the A + 1 start site) were subjected to in vitro transcription analysis with aDrosophila embryo nuclear extract. The resulting transcripts were detected by primer extension-reverse transcription analysis. (B) The core promoters of Caudal target genes contain functional DPE motifs. (ftz)fushi tarazu; (gt)giant; (h)hairy; (fkh)forkhead. In vitro transcription analysis was performed as inA.
Caudal, a conserved master regulator of the Hox gene network, is a core-promoter-specific activator. (A) Caudal activates transcription with theAntp P2 DPE motif but not with the AdML TATA element. The two reporter constructs are identical except for the TATA and DPE sequences.Drosophila S2 cells were transfected with firefly luciferase reporter constructs as well as a Caudal expression plasmid, where indicated. To normalize for transfection efficiency, cells were cotransfected with a Pol III-Renilla luciferase control plasmid and assayed for dual luciferase activity. Error bars represent the SEM. (B) TheAdh distal enhancer activates transcription with both the AdML TATA box and theAntp P2 DPE motif. The four reporter constructs are identical except for the presence or absence of the TATA, DPE, andAdh distal enhancer, as indicated. TheAdh distal enhancer contains two binding sites for the Adf-1 transcription factor, which is present in S2 cells. Transfection assays were performed with the indicated constructs as inA. Error bars represent the SEM. (C) Caudal activates transcription with theE74B DPE motif and, to a lesser extent, theAdh proximal TATA element. Transfection assays were performed with the indicated constructs as inA. Error bars represent the SEM.
Transcriptional activation by Caudal is suppressed by the presence of a BREu motif upstream of the TATA box. The two reporter constructs are identical except for the absence or presence of the AdML BREu motif immediately upstream of the TATA box, and were analyzed as in Figure 3. Error bars represent the SEM.
Caudal is a DPE-specific transcriptional activator. (A) Caudal preferentially activates transcription through the DPE in thegt core promoter. The reporter constructs contain six Caudal-binding sites upstream of thegt core promoter, which has both DPE and TATA motifs but lacks a BREu. The constructs are identical except for the mutation of the TATA and DPE motifs and the addition of the AdML BREu, where indicated. The transfection experiments were performed as in Figure 3. Error bars represent the SEM. (B) Caudal primarily activates through the DPE in the naturalftz transcriptional control region. Theftz core promoter contains both DPE and TATA motifs. The reporter constructs containftz enhancer and promoter sequences from −988 to +40 relative to the +1 start site, and are identical except for mutation of the DPE or TATA, as depicted. The transfection experiments were performed as in Figure 3. Error bars represent the SEM.
The naturalAntp P2 andScr promoters are activated by Caudal and are dependent on the DPE core promoter motif in S2 cells. (A)Antp P2 promoter. Constructs containing the wild-type and mutant versions of theAntp P2 promoter region (from −3033 to +61 relative to A + 1 in the Inr) were analyzed as in Figure 3. The activities are reported relative to the wild-typeAntp P2 promoter in the absence of cotransfected Caudal expression plasmid, which was defined to be 1. Error bars represent the SEM. (B)Scr promoter. Constructs containing the wild-type and mutant versions of theScr promoter region (from −3103 to +110 relative to A + 1 in the Inr) were analyzed as in Figure 3. The activities are reported relative to the wild-typeAntp P2 promoter in the absence of cotransfected Caudal expression plasmid, which was defined to be 1. Error bars represent the SEM.
A model for DPE-specific activation of transcription by Caudal. This model depicts a hypothetical segment of a regulatory network that comprises specific connections between activators and core promoter motifs. Caudal is a DPE-specific activator. TATA-specific activators might also exist. The ability of Caudal to function with a TATA box is further reduced by the presence of a BREu motif, which is located immediately upstream of a subset of TATA elements.
References
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