Fig. 3: Evidence for gain-of-function of periodic HOXD4 mutants in vivo.
From: An activity-specificity trade-off encoded in human transcription factors
a, Differential interference contrast (DIC) microscopy of the indicated cell lines (top). Representative fluorescence microscopy images of cell nuclei (bottom). The fusion proteins were visualized using anti-GFP immunofluorescence in fixed cells. Dashed white lines represent the nuclear contour. Scale bars, 0.4 mm (DIC microscopy) and 10 μm (fluorescence microscopy). b, Representative images of HAP1 HOXD4 wild type–mEGFP, HOXD4 AroPERFECT–mEGFP and HOXD4 AroPLUS–mEGFP nuclei after 24 h of HOXD4 expression. The fusion proteins were visualized using mEGFP fluorescence in fixed cells. The number of individual nuclei per condition is provided. Scale bar, 5 μm. a,b, The normalized signal intensity was calculated by dividing the s.d. of the mEGFP signal of each nucleus by the corresponding mean mEGFP signal. c, Granularity scores of nuclei with the corresponding mean nuclear mEGFP intensities. Data are the mean ± s.d. of n = 536 (wild-type), 565 (AroPERFECT) and 504 (AroPLUS) nuclei pooled from two independent replicates. a.u., arbitrary units. d, Principal component (PC) analysis of the RNA-seq expression profiles of parental HAP1, HOXD4-knockout and the indicated knock-in HAP1 cell lines. e, Differential expression analysis of HOXD4 AroPERFECT–mEGFP and HOXD4 AroPLUS–mEGFP versus HOXD4 wild type–mEGFP HAP1 cells. P values were determined using the Benjamini–Hochberg method. f, Western blot analysis of HOXD4–mEGFP, IFI16 and ARHGAP4 in the indicated cell lines. HOXD4–mEGFP proteins were probed with anti-GFP. HSP90 was used as the loading control. HOXD4 targets (blue dot) and non-HOXD4 targets (red dot) are highlighted. g, Schematic model of the condensate tethering system (left). Fluorescence images of ectopically expressed YFP–RNAPII CTD in live U2OS cells cotransfected with the indicated cyan fluorescent protein (CFP)–LacI-HOXD4 IDR fusion constructs (right). The dashed line represents the nuclear contour. Inserts: magnified views of the regions in the red boxes. Scale bars, 10 μm (main images) and 40 μm (inserts). h, Relative YFP signal intensity in the tether foci. Data are the mean ± s.d. of n = 50 (wild-type YFP and wild-type YFP–RNAPII CTD), 51 (AroPERFECT YFP) and 53 (AroPERFECT YFP–RNAPII CTD) nuclei pooled from two independent replicates. c,h, P values are from two-sided unpaired Student’s t-tests; NS, not significant.
