NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities, which plays essential roles in mammalian gene expression. As part of the current approaches to solve its 3D structure and study its mechanisms of action, my work deals with the expression of recombinant NuRD components in both mammalian and insect cells. More specifically, I am carrying out cotransfections with gene constructs coding for different subunits, and analysing the produced proteins by pull down assays. This way we can, on the one hand, gain information about how each component interacts with the others, and, on the other hand, aim for purifying small subcomplexes that could be structurally studied by electron microscopy, or functionally analysed.

As a first group of experiments, we studied mammalian HEK 293 cells cotransfected with different combinations of the following NuRD components: the histone binding proteins RbAp46 and RbAp48, the metastasis associated proteins MTA1 and MTA2, and the histone deacetylase HDAC1. By pull down analysis, we obtained data which suggest that RbAp46/48 interacts with both MTA1/2 and HDAC1, and that MTA1/2 binds HDAC1 as well. Even if we obtained tangible differences between given pairs of binders, we cannot rule out the presence of endogenous NuRD components interfering in the results. In this regard, using a non-mammalian expression system, such as Baculovirus infected insect cells, is expected to lessen this effect. Furthermore, it would render larger protein yields.

In conjunction, recombinant alternatives of producing the NuRD complex would be a robust approach to studying this key element of the mammalian gene expression.