Figure 6. Interplay between H4 K16ac and H2B K34ub.

(A) Top, schematic for the sequential in vitro HAT and histone ub assays. Bottom, MOF-MSL was first incubated with or without [³H]-acetyl-CoA as indicated on top. After an hour at 30°C, ATP was added to each reaction for the following histone ubiquitylation reaction. (B) MOF-MSL was first incubated with substrate, E1, E2 with or without ATP as indicated on top. After an hour at 30°C, [³H]-acetyl-CoA was added to each reaction for the following HAT reaction. In both (A) and (B), H4ac and H2Bub were detected by autoradiograph and anti-HA antibody respectively. Coomassie stained gel for nucleosomes was included. (C) In vitro HAT assays for different MOF-MSL sub-complexes as indicated on top. [³H]-acetyl-CoA was used as cofactor. H4ac was detected by autoradiograph. Immunoblot for H4 was used as the loading control. (D) Immunoblots for histone modifications using antibodies as indicated on left. Total proteins were isolated from HeLa cells after control, MSL1 or MSL3 siRNA treatment. (E) RT-PCR for cells treated with control, MSL1 or MSL3 siRNAs. Genes were indicated at bottom. Gene expression was normalized against GAPDH and presented as fold change against control siRNA treated sample, which is arbitrarily set at 1. (F–J) ChIP experiments in MSL1 or MSL3 siRNA treated cells using the indicated antibodies (on top). ChIP was quantified using the probe sets shown in Figure 5B. Signals for each experiment were normalized to 5% input. Means and standard deviations (error bars) from at least three independent experiments were presented.