Journal:

Article Title: RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells

doi: 10.1016/j.cell.2009.02.027

Figure Lengend Snippet: (A) Human RAD6 can fully complement the function of yeast Rad6 for H2B ubiquitylation and H3K4-H3K79 methylation. Yeast whole cell extracts from a wild-type strain (containing a chromosomal FLAG-H2B gene), its isogenic Δrad6 strain, and Δrad6 strains that harbor the indicated E2 expression plasmids (driven by the natural yeast Rad6 promoter) were subjected to immunoblotting with the indicated antibodies. Two different amounts of cell extracts were loaded. (B) Effects of siRNA-mediated knockdown of H2B ubiquitylation factors on histone modifications. 293T cells were treated with control, hBRE1A, hBRE1B, hRAD6A, hRAD6B and hUbcH6 siRNAs indicated. Total cell lysates were subjected to immunoblot analyses with indicated antibodies. A nonspecific band in the hUbcH6 immunoblot is indicated by an asterisk. (C) Total RNAs from siRNA-treated cells were subjected to RT-PCR analyses for mRNA levels of indicated genes.

Article Snippet: Although a concomitant decrease in H2B ubiquitylation and total RAD6 protein was only observed with hRAD6A siRNA, and not with hRAD6B siRNA, this reflects the fact that hRAD6A is much more abundant than hRAD6B in cells ( Koken et al., 1996 ).

Techniques: Methylation, Expressing, Western Blot, Knockdown, Control, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells

doi: 10.1016/j.cell.2009.02.027

Figure Lengend Snippet: (A) Analyses of purified hBRE1 complex, FLAG-hBRE1A, FLAG-hBRE1B, GST and GST-E2 proteins by Coomassie blue staining. Asterisk, degradation product. (B) Selective binding of the hBRE1 complex to hRAD6A and hRAD6B. GST pull down assays employed the purified proteins shown in (A) and bound proteins were scored by immunoblotting with indicated antibodies. (C) Intracellular binding of the hBRE1 complex to hRAD6. Total cell lysates from a FLAG-hBRE1A (RNF20) 293T cell line were incubated with M2 agarose, and bound proteins were visualized by immunoblotting with indicated antibodies. (D) Analysis of purified His-pK-HA-ubiquitin, FLAG-hE1, and His-E2 proteins by Coomassie blue staining. (E and F) Ubiquitylation of the hBRE1 complex by hRAD6. The purified hBRE1 complex was analyzed in an E3 ubiquitylation assay with indicated E2 enzymes in the presence of either 32P-lablled (E) or unlabelled (F) ubiquitin, respectively, and ubiquitylation of the hBRE1 complex was monitored by autoradiography (E) or immunoblot (F), respectively. Ub×2 indicates a ubiquitin dimer. (G) Binding of the purified hBRE1 complex containing either FLAG-hBRE1A N381 or N230 fragments (Figure 1C) to hRAD6A and hRAD6B. (H) Schematic of direct binding of the hBRE1 complex to hRAD6.

Article Snippet: Although a concomitant decrease in H2B ubiquitylation and total RAD6 protein was only observed with hRAD6A siRNA, and not with hRAD6B siRNA, this reflects the fact that hRAD6A is much more abundant than hRAD6B in cells ( Koken et al., 1996 ).

Techniques: Purification, Staining, Binding Assay, Western Blot, Incubation, Ubiquitin Proteomics, Ubiquitin Assay, Autoradiography