Extended Data Fig. 3: The GSD/brace II helix and NegC core of Chd1 antagonize nucleosome binding.
From: A competitive regulatory mechanism of the Chd1 remodeler is integral to distorting nucleosomal DNA
a. On native acrylamide gels, titration of Chd1 in the presence of competitor DNA produces two supershifted bands that represent 1:1 and 2:1 Chd1:nucleosome complexes. For these experiments, FAM-labeled 40N40 601 nucleosomes (2 nM) were incubated with increasing amounts of Chd1 (2.44–5000 nM) in the presence of DNA competitor (1 mg/ml salmon sperm DNA) and 1 mM AMP-PNP, and separated on 4.25% (60:1) native polyacrylamide gels. b. Data and fits from native gel nucleosome binding experiments to two Kd values, representing affinities for the two sides of the nucleosome. For clarity, error bars are not shown. This plot shows data and fits in AMP-PNP conditions. Number of independent replicates for wild type and each variant in AMPPNP conditions are: Chd1[wildtype], n = 5; Chd1[GSD/brace II]864-902-flex, n = 3; Chd1[GSD/brace II]864-871-flex, n = 3; Chd1[GSD/brace II]L865N/L869N, n = 4; Chd1[activator]876-881, n = 3; Chd1[NegC]884-889-flex, n = 8; Chd1[NegC]890-895-flex, n = 3; Chd1[NegC]884-902-flex, n = 3; Chd1[NegC]L886N/L889N/L891N, n = 3; Chd1[NegC]896-901-flex, n = 3; Chd1[NegC]902-907-flex, n = 4; Chd1[activator]F917N/L918N/F921N, n = 3; Chd1[activator]W932A, n = 3; Chd1[brace I]I843N, n = 3; Chd1[protrusion 2]M652Q, n = 3. c. Comparison of apparent nucleosome binding affinities for Chd1 variants in different nucleotide conditions. These 2D plots show the higher affinity apparent Kd value (Kd1,app) along y, and the lower affinity apparent value (Kd2,app) along x. The vertical dashed lines (at 7 µM) show the confidence limit for Kd2,app. Number of independent replicates for wild type and each variant in ADP and nucleotide-free conditions, respectively, are as follows: Chd1[wildtype], n = 7,9; Chd1[GSD/brace II]864-902-flex, n = 3,4; Chd1[GSD/brace II]864-871-flex, n = 7,7; Chd1[GSD/brace II]L865N/L869N, n = 4,4; Chd1[activator]876-881, n = 4,3; Chd1[NegC]884-889-flex, n = 3,7; Chd1[NegC]890-895-flex, n = 4,4; Chd1[NegC]884-902-flex, n = 3,3; Chd1[NegC]L886N/L889N/L891N, n = 3,4; Chd1[NegC]896-901-flex, n = 4,5; Chd1[NegC]902-907-flex, n = 3,3; Chd1[NegC]901-902-flexinsert, n = 3,4; Chd1[activator]F917N/L918N/F921N, n = 3,3; Chd1[activator]W932A, n = 3,3; Chd1[brace I]I843N, n = 4,3; Chd1[protrusion 2]M652Q, n = 5,3. Number of replicates for wild type and each variant in AMPPNP conditions is listed in Extended Data Fig. 3b. Affinities and standard deviations are reported in Supplementary Table 3. Data are given in Source Data File 6.
