KDM6B interacts with nucleo-adhesome components CSRP2 and TGFB1I1 to regulate EMT

Abstract

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.