Core promoter-selective function of HMGA1 and Mediator in Initiator-dependent transcription

Abstract

The factors and mechanisms underlying the differential activity and regulation of eukaryotic RNA polymerase II on different types of core promoters have remained elusive. Here we show that the architectural factor HMGA1 and the Mediator coregulator complex cooperate to enhance basal transcription from core promoters containing both a TATA box and an Initiator (INR) element but not from "TATA-only" core promoters. INR-dependent activation by HMGA1 and Mediator requires the TATA-binding protein (TBP)-associated factors (TAFs) within the TFIID complex and counteracts negative regulators of TBP/TATA-dependent transcription such as NC2 and Topoisomerase I. HMGA1 interacts with TFIID and Mediator and is required for the synergy of TATA and INR elements in mammalian cells. Accordingly, natural HMGA1-activated genes in embryonic stem cells tend to have both TATA and INR elements in a synergistic configuration. Our results suggest a core promoter-specific regulation of Mediator and the basal transcription machinery by HMGA1.

Figure 1.

Purification and identification of HMGA1 as a component of the TIC1 activity. (A) Purification scheme used. Numbers in parentheses indicate the molar KCl concentration used to elute the TIC1 activity from each resin (see also Supplemental Fig. S1). (B) Representative in vitro transcription–primer extension analysis of transcripts from TATA and TATA/INR templates in HeLa nuclear extracts (NE, lane 1) and in the system reconstituted with purified GTFs and Pol II and complemented with TIC1 fractions from the last two purification steps. (C) Analysis of the purified TIC1 Phenyl fraction by SDS-PAGE and Coomassie staining. The proteins were excised from the gel and identified by LC/ESI/MS/MS. (D,E) In vitro transcription/primer extension assays with recombinant HMGA1b were performed with supercoiled templates in the purified system containing either TFIID or TBP. Autoradiograms shown for TATA and TATA/INR are from the same gel and exposure time. E shows a quantitation (mean ± SD) of more than three independent transcription experiments normalized to the promoter activities in the absence of HMGA1b.

Figure 2.

Cooperativity of HMGA1 and Mediator in TFIID/TAF-dependent INR function. (A) In vitro transcription experiment with supercoiled TATA and TATA/INR templates. Mediator (1, 2, and 4 μL) was titrated alone (lanes 2–4) or together with 40 ng of recombinant HMGA1b (lanes 5–7) in the purified TFIID system. The histogram shows the relative transcription activities for each template (normalized to lane 1). (B) The individual and combined effects of HMGA1b (40 ng) and Mediator (2 μL) on basal transcription in the purified system were quantitated from more than three independent experiments and plotted for each supercoiled TATA and TATA/INR promoter template as relative activities (mean ± SD) normalized to promoter activities in the absence of HMGA1 and Mediator. (C–E) In vitro transcription comparing the effects of HMGA1 and Mediator on the basal activities of different supercoiled (C,D) and linear (E) core promoters in the purified system containing either TFIID or TBP. (C) The relative transcription activities and “selectivity ratio” of TATA/INR to TATA are shown. HeLa nuclear extract (NE) was used as a reference. (D,E) Promoter activities were normalized to the activity of MLP in the TFIID system in the absence of HMGA1 and Mediator (left lanes), and only the selectivity ratios (MLP/HSP70) are shown. In D, the corresponding relative transcription activites (left to right) for MLP were 1.00, 2.24, 3.27, 8.24, 1.89; and for HSP70 were 0.67, 0.93, 1.02, 1.62, 1.26. See also Supplemental Figure S2H,I.

Figure 3.

HMGA1 and Mediator counteract the negative functions of NC2 and Topo I in an INR-dependent manner. Recombinant NC2 (A) (see Supplemental Fig. S3) or Topo I (B) was added to the purified TFIID-based system in the presence or absence of HMGA1 and Mediator, as indicated. Basal transcription was analyzed from supercoiled TATA and TATA/INR promoters. Autoradiograms in each of the two panels are from the same gel and film exposure. The relative transcription signals (normalized to lane 1) and the ratio of TATA/INR to TATA signals are shown.

Figure 4.

HMGA1 is required selectively for TATA/INR but not TATA-mediated transcription in vivo. (A) TATA/INR-Luc and TATA-Luc were transfected in HEK293 cells, and the relative luciferase activities (mean ± SD) from six independent experiments (each in duplicate) are shown. The luciferase activity of TATA-Luc was arbitrarily set to 1. (B) HEK293 cells were transfected with either control (Contr.) or specific siRNA against HMGA1. Endogenous HMGA1 in whole-cell extracts was analyzed by Western blot. (C) TATA/INR-Luc, TATA-Luc, or ACTB-Luc reporters were transfected in HEK293 cells with a control siRNA (black bars) or the HMGA1-specific siRNA (open bars). The relative luciferase activities are shown (as in A). TATA-Luc activity (left panel) and ACTB-Luc activity (right panel) in cells transfected with the control siRNA were set arbitrarily to 1. (D) Total mRNA from HEK293 cells transfected with ACTB-Luc (lanes 1–6) and either TATA/INR-Luc (lanes 1–3) or TATA-Luc (lanes 4–6) and the indicated siRNAs was analyzed by primer extension with the Luc 24mer primer. Lane 7 is a control reaction with mRNA from mock-transfected cells. The position of correctly initiated transcripts is indicated for each promoter construct (length of 172 nucleotides [nt] for ACTB-Luc and 128 nt for TATA and TATA/INR-Luc). The top and bottom autoradiograms are from the top and bottom parts of the same gel; the bottom autoradiogram is from a longer X-ray film exposure time. See also Supplemental Figure S4 for an analysis of a different TATA/INR core promoter.

Figure 5.

HMGA1 interacts with TFIID and Mediator, but not CDK8, in human cells. (A) Western blot analysis of total HMGA1 in normal HEK293 cells (−) and a derivative clonal cell line that was stably transfected with HA-HMGA1b (+). Positions of endogenous HMGA1 and ectopic HA-HMGA1b proteins are indicated. An antibody to β-actin was used as loading control. (B,C) Whole-cell extracts from HEK293 cells and HEK293 cells stably expressing HA-HMGA1b (described above) were adjusted to 175 mM KCl and immunoprecipitated with anti-HA antibody resin in the presence or absence of ethidium bromide (EB), as indicated. The Western blot was probed with antibodies to the indicated TFIID and Mediator subunits. NC2α served as the negative control. (D) HeLa cell nuclear extracts were immunoprecipitated with a MED1 antibody or mock-immunoprecipitated with goat IgG (Mock), and associated proteins were analyzed by Western blot with the indicated antibodies. Similar results were obtained with HEK293 cells (Supplemental Fig. S5).

Figure 6.

The HMGA1 acidic C-tail domain is required for interaction with TFIID and Mediator and for stimulation of INR-dependent transcription. (A) Scheme of HMGA1b wild-type structure, including AT hooks (AT) and acidic tail (C tail), and deletion mutants used for immunoprecipitation experiments below. (B) HEK293 cells were mock-transfected (−) or transiently transfected with HA-HMGA1b wild type (1–96) or the indicated deletion mutants. Whole-cell extracts (input) were immunoprecipitated with anti-HA antibody (IP: HA) and analyzed by Western blot with the indicated antibodies. (C,D) In vitro transcription was performed with the indicated core promoter constructs (linear form) in the purified TFIID-based system complemented with Mediator and different amounts (20, 40, and 60 ng in C; and 35, 40, and 45 ng in D) of either wild-type HMGA1b (wt) or a deletion mutant 1–81 (ΔC) that lacks the acidic C-tail domain (purified proteins are shown in Supplemental Fig. S6). Relative transcription levels were normalized to the signals in the absence of HMGA1/Mediator (shown in lanes 1).