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. 2012 Mar 2;287(10):7134-45.
doi: 10.1074/jbc.M111.291385. Epub 2012 Jan 11.

Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition

E-Jean Tan  1 , Sylvie Thuault, Laia Caja, Tea Carletti, Carl-Henrik Heldin, Aristidis Moustakas
Affiliations

Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition

E-Jean Tan et al. J Biol Chem. .

Abstract

Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor β activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.

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Figures

FIGURE 1.
FIGURE 1.
HMGA2 induces Twist expression during EMT. A, quantitative RT-PCR analysis of Snail and Twist mRNA levels in parental NMuMG cells and a cell clone of NMuMG expressing constitutively human HMGA2 (HMGA2-NMuMG). Each bar represents mean ± S.D. values from triplicate samples. B, immunoblots of Snail and Twist protein levels in cytosolic and nuclear fractions of cells described in A. PARP-1 and α-tubulin serve as markers for the nuclear and cytosolic fraction respectively. C, D, and F, luciferase reporter assay of Twist promoter constructs in HepG2 cells transiently transfected with pcDNA3 empty vector or HA-HMGA2 (C); stimulated with 5 ng/ml TGFβ1 for the indicated time period (D); transiently transfected with FLAG-Smad3, FLAG-Smad4, and/or HA-HMGA2 plasmids and stimulated or not with 5 ng/ml TGFβ1 (F). Each bar represents mean ± S.D. values of normalized luciferase data from triplicate samples. E, quantitative RT-PCR analysis of Twist mRNA levels in parental NMuMG cells, untreated or treated with 5 ng/ml TGFβ for 2, 4, 8, 12, and 24 h. Each bar represents mean ± S.D. values from triplicate samples.
FIGURE 2.
FIGURE 2.
HMGA2 binds directly to the Twist promoter. A, schematic diagram of the murine Twist promoter (top panel). Nucleotide numbers were assigned relative to the TSS, and two putative TATA boxes are denoted by white bars. Fragments a, b, and c indicate PCR amplicons analyzed in panel G with ChIP. Luciferase reporter assays of a deletion series of Twist promoter constructs in HepG2 cells transiently transfected with pcDNA3 or HA-HMGA2 plasmid (bottom panel). B and C, biotin-labeled probes spanning −95 to +23, +7 to +109, and +93 to +198 (B), and probes spanning +7 to +37, +38 to +65, +66 to +87, and +88 to +109 (C), were used in DNAP experiments using extracts of HepG2 cells transiently transfected with HA-HMGA2 plasmid. D, nucleotide sequences of wild-type (WT) and mutant (M1–3) probes corresponding to the +66 to +87 region of the Twist promoter. A line indicates unaltered sequences. Binding of HMGA2 to WT or mutant probes was assessed by DNAP experiments using extracts of HepG2 cells as described in B. Probe +7/+37 is not bound by HMGA2 and serves as an additional negative control. TCL, total cell lysates in B–D. E, luciferase reporter assays of wild type or mutants (M2 and M3) −95/+209 Twist promoter constructs in HepG2 cells transiently transfected with pcDNA3, HA-HMGA2 wt, or HA-HMGA2ΔC plasmids. F, luciferase reporter assays of a deletion series of Twist promoter constructs based on the −95/+209 Twist promoter in HepG2 cells transiently transfected with pcDNA3 or HA-HMGA2 plasmid. Each bar represents mean ± S.D. values of normalized luciferase data from triplicate samples. G, ChIP assays in HMGA2-NMuMG cells were used to analyze binding of HMGA2 protein along the Twist promoter using anti-HMGA2 or control IgG antibodies. Quantitative PCR was performed, and values are expressed as percentage of input DNA. Each bar represents mean ± S.D. values from triplicate samples. The PCR amplicons, −73/+77, −23/+84, and −1190/−1059, used are denoted as a, b, and c in panel A.
FIGURE 3.
FIGURE 3.
Twist depletion in HMGA2-NMuMG cells causes a mild re-epithelialization. A, quantitative RT-PCR analysis of Twist, Snail, Slug, ZEB1, ZEB2, and E-cadherin mRNA levels; and B, immunoblot of Twist, Snail, ZEB1, and ZEB2 proteins in NMuMG-m, HMGA2-NMuMG cells (HMGA2, −), and its derivatives constitutively expressing shRNA against Twist (HMGA2-shTwist #25 and #28) or control lacZ (HMGA2-shlacZ). NMuMG-m is a sub-clone of parental NMuMG, which exhibits a highly polarized epithelial morphology, and serves as a mock NMuMG-transfected clone for HMGA2-NMuMG cells (14, 15). Each bar represents mean ± S.D. values from triplicate samples. C, cellular morphology of parental NMuMG cells, HMGA2-NMuMG cells, and its derivatives described in A. Bar, 10 μm.
FIGURE 4.
FIGURE 4.
Protein marker analysis in HMGA2-NMuMG cells after Twist depletion. Immunostaining for E-cadherin, ZO-1, and fibronectin and phalloidin staining (actin) in HMGA2-shTwist clones, as described in Fig. 3A. Bar, 20 μm.
FIGURE 5.
FIGURE 5.
Twist and Snail depletion in HMGA2-NMuMG cells causes stronger epithelial reversion. A, quantitative RT-PCR analysis of Twist, Snail, Slug, ZEB1, ZEB2, and E-cadherin mRNA levels; and B, immunoblots of Twist, Snail, ZEB1, and ZEB2 proteins in NMuMG-m and HMGA2-NMuMG cells (HMGA2, −), and cell clones of HMGA2-shSnail constitutively expressing shRNA against Twist (HMGA2-shSnail/Twist #49 and #50) or its control HMGA2-shSnail/lacZ. Each bar represents mean ± S.D. values from triplicate samples. C, cellular morphology of parental NMuMG, HMGA2-shlacZ, and HMGA2-shSnail/Twist clones described in A. Bar, 10 μm.
FIGURE 6.
FIGURE 6.
Twist and Snail depletion in HMGA2-NMuMG cells causes tight junction reassembly. Immunostaining for E-cadherin, ZO-1, and fibronectin and phalloidin staining (actin) in HMGA2-shSnail/Twist clones, as described in Fig. 5A. Bar, 20 μm.
FIGURE 7.
FIGURE 7.
Diagram of the molecular pathway that leads to EMT downstream of TGFβ and HMGA2, as analyzed in this study. TGFβ, via Smads induces expression of HMGA2. HMGA2 then binds to the promoters of Snail and Twist and induces their expression. The Snail gene also receives additional inputs from TGFβ, such as ERK MAPK signaling. The five pro-EMT transcription factors (boxed) positively cross-regulate each other to elicit nuclear reprogramming that leads to EMT. Snail and Twist cross-regulate expression of each other albeit weakly (dotted arrow). Snail regulates expression of Slug, ZEB1, and ZEB2, whereas Twist regulates expression of Slug and ZEB1. The possible cross-regulation between ZEB1, ZEB2, and Slug was not examined in this study.
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