TBX2 bind to the <i>E-cadherin</i> promoter <i>in vivo and</i> represses <i>E-cadherin</i> transcription.

<p>(A) Schematic of the <i>E-cadherin/CDH1</i> promoter depicting the location of potential TBX2 binding sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041355#pone.0041355-Redmond1" target="_blank">[10]</a>: half T-site (black box) and InR = Initiator element (hatched box), and of ChIP primers used in (B). (B) ChIP analysis shows <i>in vivo</i> binding of exogenous TBX2 to the most proximal region of the endogenous <i>E-cadherin</i> gene promoter in HC11 mammary epithelial cells. DNA derived from sheared chromatin fragments from HC11+vector and HC11+TBX2 was immunoprecipitated with two antibodies specific to TBX2 (#1 = Millipore AB4147; #2 = SC-17880x), an antibody specific to acetyl Histone 3, or normal rabbit IgG and quantified by semi-quantitative PCR. As a control, <1% of input chromatin was used in the PCR analysis. The bar graph on the bottom panel shows a quantification of the TBX2-specific ChIPs for the proximal (−131/+61) <i>E-cadherin</i> promoter as a function of the percentage of input chromatin. (C) Transient reporter assays of HC11 cells transiently co-transfected with a human <i>E-Cadherin</i> promoter (−108 to +125) luciferase reporter construct (pEcad-luc) in combination with pCDNA3 vector (basal) or increasing concentrations of pCDNA3-TBX2 (+TBX2), as indicated. One representative experiment of n = 3 biological replicates is shown; P-value: *<i>p<0.05</i> (triplicate samples; Student <i>t</i>-test).