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. 2010 Aug 17;21(2):275–289. doi: 10.1038/cr.2010.118

Figure 3.

Figure 3

TWIST recruits the Mi2/NuRD/MTA2 complex to E-cadherin promoter and depends on MTA2 to repress E-cadherin expression. (A) The proximal region of the E-cadherin promoter contains three TWIST-binding E-boxes. In the ChIP assay, PCR with P51 and P32 primers was used to amplify the region with these E-boxes. PCR with primers P3 and P4 in the 3′ region of the E-cadherin gene, which was more than 100 kb downstream from the indicated E-boxes, served as negative control in the ChIP assay. (B) ChIP assays using F-TWIST and F-vector HEK293 cells, Flag antibody or control IgG and primer pairs of P51/P32 and P3/P4 for E-cadherin gene. (C) ChIP-re-ChIP assays for the E-box region of the E-cadherin gene. The first-step immunoprecipitation was performed with Flag antibody and cross-linked and sonicated chromatin of F-TWIST and F-vector HEK293 cells. The second-step immunoprecipitation was performed with the eluted materials from the first step and with antibodies against Mi2, HDAC2 and MTA2 as indicated. IgG served as a negative control in the second step ChIP. (D, E) Generation of stable 4T1 cell pools with lentivirus-mediated expression of Twist shRNA (shTwist-74), Mta2 shRNA (shMta-54 and -56), RbAp46 shRNA (shRp-20 and -21) and non-targeting shRNA (shCtrl). Panel D shows efficient knockdown of Twist and the knockdown did not affect RbAp46 (Rp) and Mta2. Panel E shows efficient knockdown of Mta2 and RbAp46 proteins, and knockdown of any one of two proteins did not affect the other one (left panel) and also did not affect Twist (right panel). (F) Knockdown of Twist, Mta2 or RbAp46 in 4T1 cells increases E-cadherin promoter activity. 4T1 cell pools carrying non-targeting shRNA (control) and targeting shRNAs for Twist, Mta2 and RbAp46 as indicated were transfected with 100 ng of the E-Cad-promoter-Luciferase reporter plasmid and 30 ng of β-gal expression plasmids in 24-well plates. Luciferase activity was assayed 40 h later, normalized to β-gal activity and presented as mean ± SD, n = 3. (G) TWIST represses endogenous E-cadherin expression. HEK293 cells with F-TWIST and F-vector were set up in three groups. Groups 1 and 2 were treated with vehicle (lane 1 and 4) and DOX for 2 days to induce F-TWIST (lanes 2 and 3) and Flag (lanes 5 and 6). Group 3 was treated with DOX for 2 days, and then DOX was removed and cells were cultured for 7 more days (lanes 3 and 6). Western blot analyses were performed to analyze E-cadherin, F-TWIST and β-actin. (H) Western blot analysis of MTA2 in puromycin-selected stable F-TWIST HEK293 cells infected by lentiviruses with a non-targeting control shRNA and five different shRNAs against human MTA2 mRNA. The shRNA-MTA2-77 lentivirus successfully reduced both MTA2 and MTA1 proteins. (I) MTA2 knockdown blocks TWIST-mediated suppression of E-cadherin expression. shRNA control and shRNA-MTA2-77 cells were treated with vehicle or DOX to induce F-TWIST for 3 days before western blot was performed to assay E-cadherin, F-TWIST and β-actin.