Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription

Abstract

Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.

Keywords: Core promoter-selective transcription activation; Gene regulation; RNA polymerase II; TATA box; TATA-binding protein.

Figure 1. An INR cannot stimulate transcription from the human ACTB promoter in vivo

A. Schematic diagrams of luciferase reporters containing the natural ACTB promoter (−120/+100 bp) or the natural HSV TK promoter (−113/+57bp) and derivatives [+INR] containing a consensus INR sequence (CTCATTCT) indicated in bold at the transcription start site (bent arrow; +1). The TATA box sequence of ACTB is TATAAA and that of HSV TK is TATTAA. The upstream activating sequence (UAS) of ACTB contains two essential elements: a CCAAT box and a CArG box. The UAS of HSV TK promoter contains two essential GC-box elements. B. Relative luciferase activities of the constructs in panel A transfected into HEK293 cells. Activities of ACTB-Luc and HSV-TK-Luc lacking an INR (gray bars) were arbitrarily set to 1. Results are the means ±S.D. of three independent experiments. C. Primer extension analyses of RNAs transcribed from the ACTB-Luc or ACTB[+INR]-Luc reporters transfected in HEK293 cells (top panel) or after in vitro transcription in a HeLa nuclear extract (bottom). Correctly initiated transcripts are indicated by “+1”.

Figure 2. The INR stimulates transcription activation by different types of activation domains in vivo

A. Schematic diagram of promoter-luciferase reporters and Gal4-fusion activators. Reporters contain five Gal4 binding sites upstream of TATA (T), TATA/INR (T/I) or INR (I) core promoters. Gal4(1–147) is the yeast Gal4 DNA binding domain (amino acids 1–147). The activation domains of SP1 (132–243), CTF1 (399–499) and VP16 (411–490) were fused to Gal4(1–147) to generate Gal4-SP1, Gal4-CTF1 and Gal4-VP16 fusion activators, respectively. B. The reporters above were co-transfected with Gal4(1–147), Gal4-SP1, Gal4-CTF1 or Gal4-VP16 in HEK293 cells. Luciferase activities are relative to the activity of the promoter-less pGL3 luciferase vector, which was arbitrarily set to 1, and are the means ±S.D. of three independent experiments. The basal activities without any activator are shown in supplemental Fig. S1A. Bottom panels show autoradiograms of representative primer extension analyses indicating correctly initiated transcripts (+1) in transfected cells, including an endogenous cellular transcript used as an internal control (ctrl). The autoradiogram for Gal4-VP16 resulted from a shorter X-ray film exposure.

Figure 3. The UAS of ACTB is composed of a CCAAT box and a CArG box that synergistically activate TATA-dependent but not INR-dependent transcription

Top, are diagrams of the reporters containing the wild type (WT) UAS of ACTB (−120 bp to −40 bp) or mutated (mut.) derivatives upstream of heterologous TATA (T), TATA/INR (T/I) or INR (I) core promoters. The specific nucleotides substituted in the CCAAT-box (mut.CCAAT), CArG-box (mut.CArG) or both boxes (mut.Both) are indicated with asterisks. Bottom, are the relative luciferase activities of wild type and mutant reporters transfected into HEK293 cells (n ≥ 3 independent experiments). The Luc activities are relative to that of the promoter-less pGL3 luciferase vector, which was arbitrarily set to 1. Note the two different scales for the Rel. Luc Activities of WT and mutated ACTB UAS reporters. A representative primer extension analysis is also presented showing the correctly initiated transcripts (closed arrowhead; +1). A low level of incorrectly initiated transcription is seen mostly in the INR (I) lanes (open arrowhead). An endogenous cellular transcript served as internal control (ctrl), as in Fig. 3.

Figure 4. Transcription activation by the ACTB UAS is unidirectional on TATA but bidirectional on a composite TATA/INR core promoter

The top part shows the luciferase reporters containing the UAS of ACTB (−120 to −40 bp) inserted upstream of heterologous TATA, TATA/INR or INR core promoters in forward or reverse orientation. The lower part shows the luciferase activities of the above reporters in transfected HEK293 cells. Luciferase activities are the means of three independent experiments. Primer extension analyses of correctly initiated Luc transcripts (+1), including an internal control (ctrl) transcript (as in Figs. 2 and 3), are shown at the bottom.

Figure 5. The TATA-binding activity of TBP is required in vivo for the TATA-containing ACTB and MYC promoters but not for the TATA-less CDKN1A promoter

A. Western blot analyses of endogenous TBP knockdown in Hela S3 and HeLa S3+T210K cell lines. The top stripe was probed with a TBP antibody. Endogenous TBP wild type (endo.WT) and HA-tagged TBP-T210K mutant (T210K) proteins are indicated with arrowheads. The bottom stripe was probed with a TAF5/TAF100 antibody. No siRNA (lanes 1 and 4), a control siRNA (lanes 2 and 5) or the TBP 3′-UTR siRNA were transfected into both cell lines, as indicated. TBP knockdown under the conditions used (see Materials and Methods) did not affect expression of TAFs or other proteins (see also Supplemental Fig. S4). B. Expression levels of ACTB pre-mRNA, and ACTB, MYC and CDKN1A mRNAs were quantitated by RT-qPCR in HeLa S3 (black bars) and HeLa S3+T210K (gray bars) cells after transfection with the TBP siRNA (si-TBP) and are relative to the levels of expression of each gene transcript in cells transfected with the control siRNA (si-Ctrl; white bars), arbitrarily set to 1. The asterisks indicate significant differences relative to si-Ctrl transfected cells (P<0.05). C. Relative activities of promoter-luciferase constructs containing the natural ACTB or CDKN1A promoters or UAS-ACTB-TATA. Luciferase reporters were transfected into Hela S3+T210K cells in the conditions of mock or knockdown of endogenous TBP. Luciferase activity was averaged from three independent experiments. Luciferase activity from si-Ctrl samples was arbitrarily set to 1.

Figure 6. ACTB promoter activity requires SRF but not NFY or SP1

A. Relative luciferase activity from the natural ACTB promoter-luciferase construct (see Fig. 1A) in cells transfected with control (Ctrl), SRF, NFYA, NFYB or SP1 siRNAs. Luciferase activity was normalized to total protein. Luciferase activity in cells transfected with the siRNA Ctrl was arbitrarily set to 1. Results are the means from three independent experiments. Asterisks indicate significant differences (P<0.05) relative to Ctrl. B. Western blot analysis of SRF, NFYA, NFYB and SP1 proteins in extracts of cells transfected with the control siRNA (si-Ctrl) and specific siRNAs as indicated.