Fig. 4

The degenerated histone-binding motif of the CW domain of MORC2 binds an Arg-rich surface on the ATPase module. a Amino acid sequence alignment of the CW domains of MORC2, lysine-specific demethylase 1B (KDM1B) and MORC3, shows that MORC2 lacks the ‘floor’ Trp residue important for H3K4me3 coordination by the other proteins. The sections of the MORC domain structure schemes marked with a line represent the regions resolved in crystal structures. ‘Zn’ marks the cysteine residues involved in zinc coordination. Residue numbers refer to human MORC2. b Details of the MORC2 ATPase–CW interaction. Key residues mentioned in the text are shown in stick representation and polar contacts (3.2 Å or less) are represented by dotted lines. c–e Weakening the ATPase–CW interaction in MORC2 hyperactivates HUSH-mediated transgene silencing. Time-course of the transgene re-repression by R252W and R266A MORC2 variants in the MORC2-knockout HeLa reporter clone (c). The bar chart shows the relative GFP fluorescence level (FACS-derived geometric mean) of complemented cells compared to the untreated cells (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant, unpaired t-test compared to wild-type data, n = 3). Error bars represent the mean ± standard deviation from three biological replicates. Example FACS plots show that the R252W and R266A variants enhance the overall degree of GFP reporter repression relative to wild-type MORC2 (d). Shown are the data from Day 14 post-transduction: the GFP reporter fluorescence of the HUSH-repressed clone is in gray; the MORC2 knockout is in green; the MORC2 knockout transduced with exogenous MORC2 variants is in orange. Western blot validation of expression of the MORC2 variants (e)