Journal: bioRxiv

Article Title: Periphilin self-association underpins epigenetic silencing by the HUSH complex

doi: 10.1101/2019.12.18.881300

Figure Lengend Snippet: Periphilin and TASOR form a 2:1 complex required for HUSH function. ( A ) Crystal structure of the Periphilin-TASOR core complex. The Periphilin fragment (residues 292-367, light/dark grey) forms a homodimer of helical hairpins. The TASOR fragment (residues 1014-1095, rainbow colors) wraps around the Periphilin dimer, adding an α -helix to each Periphilin hairpin to form two helical coiled coils. Insets show close-up views of the Periphilin-TASOR interfaces (“i”, “iii”) and the Periphilin dimer interface (“ii”). Residues forming key contacts and mutations designed to disrupt Periphilin-TASOR complex formation are labeled. ( B ) SEC-MALS of Periphilin-TASOR core complex. The molecular weight calculated from light scattering data is consistent with a 2:1 complex in solution. ( C ) Repression of a lentiviral GFP reporter in Periphilin KO cells complemented with Periphilin mutants designed to inhibit Periphilin-TASOR complex assembly. Reporter expression was monitored over 21 days by flow cytometry. The log 10 (GFP fluorescence) data for live cells were converted to percent repression activity with wild-type HeLa set at 100% and Periphilin KO cells set a 0% repression (see Methods). ( D ) Immunofluorescence microscopy of Periphilin KO cells transduced with Periphilin mutants affecting Periphilin-TASOR complex assembly. Cells were fixed 4 days post-transduction and stained with anti-Periphilin antibody (magenta) and DAPI (grey, insets). Scale bar, 10 µm.

Article Snippet: The SEC system was coupled to a multi-angle light scattering (MALS) module (DAWN-8+, Wyatt Technology).

Techniques: Labeling, Molecular Weight, Expressing, Flow Cytometry, Fluorescence, Activity Assay, Immunofluorescence, Microscopy, Transduction, Staining