Supplementary Figure 6: Characterization of the Inducible dn-remodeler system.

(a) Top, alignment of hBRG1, mouse Chd4, and hSNF2H ATPase domain region containing a conserved lysine (K) residue essential for catalytic activity. Bottom, schematic representation of dominant-negative remodeler constructs. Each remodeler contains either a single or triple FLAG tag at its N- or C-terminus for the monitoring of expression. Highlighted is the mutation of the conserved lysine within the ATPase domain to either arginine (R) or cysteine (C). (b) Schematic of tetracycline-regulated expression system. Each dominant-negative remodeler construct was stably transfected into cells containing the tetracycline transactivator (tTA) system. The tTA construct is positioned downstream of a tetracycline (Tet) response element (TRE) bound to a minimal CMV promoter and is itself regulated by the tTA protein (Vp16-TetR) providing autoinducible control. (c) Western blot analysis of dominant-negative (dn) protein expression derived from whole cell extracts of cultured cells in the presence or absence of tetracycline (Tet). Each remodeler was FLAG-tagged at either the N- or C-terminus and detected using a FLAG-specific antibody. As a positive control, the expression of tetracycline transactivator (tTA) was analyzed (only expressed in the absence of Tet). Actin was analyzed to determine equal loading. (d) mRNA expression of dominant-negative remodelers in the presence (+Tet, white bars) or absence of tetracycline (-Tet, black bars). Expression is shown relative to the +Tet condition (no expression) with normalization to Actin expression. Error bars represent the SEM of three independent replicates. (e-g) Effect of dn-remodeler expression on levels of alternate remodeling proteins. Western blot analysis of selected remodeler expression levels with activation of alternate dn factors. Nuclear extracts from cells either expressing (- tet), or not expressing (+tet, 48 hr.), FLAG-tagged versions of dnBrg1, dnSnf2h, and dnChd4 were tested for effects on the other remodeling proteins. Input, 15 μg of IP nuclear extract; western blots were developed with antibodies specific to FLAG, Brg1, Chd4, Snf2h, the tet regulator, or GAPDH as loading control. Black arrows indicate position of size markers; blue arrows indicated position of factor tested. (e) Cells expressing dnCHD4; Brg1 and Snf2h levels are unaffected. (f) Cells expressing dnBrg1; Chd4 and Snf2h levels are unaffected. Strong induction of the tet regulator is also shown at the top of the panel. (g) Cells expressing dn Snf2h; Brg1 and Chd4 levels are unaffected.