Early Growth Response 1 (EGR1) is a stress response transcription factor with multiple tumour suppressor roles in breast tissue, whose expression is often lost in breast cancers. We have previously shown that the breast cancer oncogene TBX2 (T-BOX2) interacts with EGR1 to co-repress EGR1-target genes including the breast tumour suppressor NDRG1. Here, we show the mechanistic basis of this TBX2 repression complex. We show that siRNA knockdown of TBX2, EGR1, Heterochromatin Protein 1 (HP1) isoforms and the generic HP1-associated corepressor protein KAP1 all resulted in growth inhibition of TBX2-expressing breast cancer cells. We show that TBX2 interacts with HP1 through a conserved HP1-binding motif in its N-terminus, which in turn leads to the recruitment of KAP1 and other associated proteins. Mutation of the TBX2 HP1 binding domain abrogates the TBX2-HP1 interaction and loss of repression of target genes such as NDRG1. Chromatin-immunoprecipitation (ChIP) assays showed that TBX2 establishes a repressive chromatin mark, specifically H3K9me3, around the NDRG1 proximal promoter coincident with the recruitment of the DNA methyltransferase DNMT3B and histone methyltransferase (HMT) complex components (G9A, Enhancer of Zeste 2 (EZH2) and Suppressor of Zeste 12 (SUZ12)). Knockdown of G9A, EZH2 or SUZ12 resulted in upregulation of TBX2/EGR1 co-regulated targets accompanied by a dramatic inhibition of cell proliferation. We show that a generic inhibitor of HMT activity, DzNep, phenocopies expression of an inducible dominant negative TBX2. Knockdown of TBX2, KAP1 or HP1 inhibited NDRG1 promoter decoration specifically with the H3K9me3 repression mark. Correspondingly, treatment with a G9A inhibitor effectively reversed TBX2 repression of NDRG1 and synergistically downregulated cell proliferation following TBX2 functional inhibition. These data demonstrate that TBX2 promotes suppression of normal growth control mechanisms through recruitment of a large repression complex to EGR1-responsive promoters leading to the uncontrolled proliferation of breast cancer cells.
Crawford, N. T., McIntyre, A. J., McCormick, A. J., D'Costa, Z. C., Buckley, N. E., & Mullan, P. B. (2019). TBX2 interacts with heterochromatin protein 1 to recruit a novel repression complex to EGR1-targeted promoters to drive the proliferation of breast cancer cells. Oncogene. https://doi.org/10.1038/s41388-019-0853-z