Fig. 4.

H2B-uLL abrogates H3K79 and H3K4 methyltransferase activity but not H2B-Ub–dependent chromatin compaction. (A) ³H-SAM hDot1L methyltransferase assays using unmodified (lane 1), H2B-Ub (lane 2), and H2B-uLL (lane 3) nucleosomes. H2B-Ub and H2B-uLL were prepared semisynthetically and contained isopeptide linkages between H2B-K120 and the ubiquitin. Nucleosomes were visualized by native PAGE followed by Sybr Gold staining (Top panel), and ³H-methyl incorporation was probed by fluorography (Middle panel). Quantification of methylation was performed by filter binding assays followed by liquid scintillation counting and reported as the direct readout (in cpm). Error bars, s.e.m. (n = 3). (B) In nucleo H3K79 methylation levels subsequent to semisynthetic incorporation of an H2B-Ub or H2B-uLL construct into native chromatin. Ub and uLL were incorporated into chromatin via protein transsplicing, and H3K79me2 levels were monitored (Middle panel) by Western blotting. For each sample, H3K79me2 levels were first adjusted relative to the total H3 signal within the sample (Top panel). All adjusted H3K79me2 signals were then normalized to the adjusted H3K79me2 level of the H2B-Ub sample (Bottom panel). Note that lane 1 (labeled H2B) refers to nuclei from nontransfected control cells. Error bars, s.e.m. (n = 3). (C) ³H-SAM methyltransferase assays using ySet1C. Methyltransferase assays using H2B- (lane 1), H2B-Ub– (lane 2), and H2B-uLL– (lane 3) containing nucleosomes were performed. Nucleosomes were visualized by native PAGE followed by Sybr Gold staining (Top panel), and ³H-methyl incorporation was probed by fluorography (Middle panel). The extent of ³H-methyl incorporation was quantified by densitometry (Bottom panel). Error bars, s.e.m. (n = 3). (D) Homo-FRET assay schematic to probe chromatin compaction. The 12-mer nucleosomal arrays containing a fluorescein labeled H2A compact upon addition of Mg²⁺. Compaction correlates with a lower SSA signal due to homo-FRET. (E) Nucleosomal array compaction as a function of Mg²⁺ for H2B- (gray line), H2B-Ub– (green line), and H2B-uLL– (red dashed line) containing 12-mer arrays. Error bars, s.e.m. (n = 3).