FIGURE 1:

Histone H3.3 is enriched in nucleoli before being incorporated into chromatin. (A) Diagram of the inducible transgene drawn to scale. Cherry-lac repressor allows the transgene integration site to be visualized. Transcription is induced from the minimal CMV promoter by the activators Cherry-tTA-ER (+)4-OHT and rtTA (+)Dox. The transcribed RNA encodes CFP fused to a peroxisomal targeting signal (SKL). The RNA is visualized by YFP-MS2, which binds to the stem-loops in the transcript. The 3′ end of the transcription unit is composed of the intron 2 splicing unit from the rabbit β-globin gene. The recruitment of YFP-tagged factors to the array can be monitored by coexpression with the DNA- and RNA-binding proteins. (B) Diagram of the H3.3 constructs expressed as YFP- or GST-fusion proteins in the recruitment and in vitro binding assays (Figure 3D). The amino acid differences between H3.3 and H3.2/H3.21 are shown in red. The red asterisks in the 4-PTM construct represent K37A, R42A, R49A, and R52A. (C) Localization of H3.3-YFP, expressed in U2OS 2-6-3 cells for 18 (a–d) and 48 h (e–h), in relation to the activated transgene array, marked by Cherry-tTA-ER. Localization of H3.3 N-tail-αN-YFP expressed for 18 (i–l) and 48 h (m–p). Arrows indicate the transgene array, and arrowheads indicate nucleoli. Yellow lines in enlarged merge insets (c, g, k, and o) show the path through which the red and green signals were measured in the intensity profiles (d, h, l, and p). Asterisks mark the start of the measured line. Scale bar, 5 μm, 1 μm (enlarged inset). (D) Percentage of transcriptionally activated cells with enrichment of H3.3-YFP and H3.3 N-tail-αN-YFP at the active transcription site and in nucleoli 18 and 48 h posttransfection. For each time point, 100 cells were counted from three independent transfections. SDs are shown in the form of error bars; p values were calculated using the unpaired t test.