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. 2020 Apr 15;6(16):eaaz0356. doi: 10.1126/sciadv.aaz0356

Fig. 3. PHF20L1 exerts transcriptional repressive activity by interacting with PRC2 and the NuRD complex.

Fig. 3

(A) Immunopurification and mass spectrometry analysis of PHF20L1-associated proteins in HEK293T and MDA-MB-231 cells. The eluates were resolved by SDS-PAGE and silver-stained, and the bands were retrieved and analyzed by mass spectrometry. (B) Cellular lysates from MDA-MB-231 cells were immunoprecipitated with antibodies against FLAG in the presence or absence of DNase. (C) Association of PHF20L1 with PRC2 and NuRD in HEK293T, MDA-MB-231, and Hs 578T cells. Whole-cell lysates were prepared, and Co-IP was performed. (D) Molecular interaction between PHF20L1 and PRC2 or NuRD subunits. GST/His pull-down assays using bacterially expressed GST/His-fused proteins and in vitro transcribed/translated proteins are shown as indicated. (E and F) GST pull-down assays with GST-fused truncated EZH2 or MTA proteins and in vitro–transcribed/translated PHF20L1. (G) Mapping the interface in PHF20L1 for the interaction between PHF20L1 and PRC2 or NuRD by GST pull-down assays with GST-fused PHF20L1 domain constructs and in vitro–transcribed/translated PRC2 and NuRD subunits. (H) Mapping the interface in PNID for the interaction between PHF20L1 and PRC2 or NuRD by GST pull-down assays with GST-fused PNID domain constructs and in vitro–transcribed/translated EZH2 and MTA1/2. (I) PHF20L1 has intrinsic transcription repressive activity. HEK293T cells were transfected with the indicated plasmids, and Gal4 luciferase reporter activity was measured. (J) Identification of the essential domains required for the transcriptional repressive activity of PHF20L1. The PHF20L1 deletions fused to the C terminus of Gal4 DNA binding domain were transfected into HEK293T cells, and Gal4 luciferase reporter activity was measured. (K) Effect of depletion of EZH2 or MTA1 on PHF20L1 repressive activity. HEK293T cells were transfected as indicated constructs along with siRNAs against EZH2 or MTA1 for 48 hours, and Gal4 luciferase reporter activity was measured. All error bars represent means ± SD of triplicate measurements that have been repeated three times with similar results. Two-tailed unpaired t test, *P < 0.05 (I to K).