We next investigated whether the interaction between the BPTF-containing NURF complex and the Smads is direct or indirect using pull-down assays. Experiments with bacterially expressed GST-Smad2 or GST-Smad 3 showed an interaction to a degree between recombinant NURF complex and each of the Smad transcription factors, but not the GST or GST-βcatenin controls (Figure 5C). Smad transcription factors are composed of functional domains at the N-terminus (MH1 domain) and C-terminus (MH2 domain). The N-terminal+linker and C-terminal regions of Smad2 were used in similar pulldown experiments. We observed that recombinant NURF complex interacts specifically with the MH2 domain of Smad2 (Figure 5C). This interaction was also observed for the Bptf and Snf2l components of native NURF complex from crude ES cell nuclear extracts (Figure 5C). We also confirmed the reported interaction of the C-terminal MH2 domain of the R-Smads with the co-regulator CBP (Figure 5C) [57]. Hence, our results suggest that NURF, like p300 and CBP maybe recruited to the promoters of TGFβ responsive genes through direct interactions with the Smad transcription factors.
