Journal: Molecular cell

Article Title: The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation Through an Interaction with Rad6

doi: 10.1016/j.molcel.2016.10.008

Figure Lengend Snippet: (A, B) Western blot analysis was performed on rtf1Δ and rtf1Δ paf1Δ ctr9Δ cdc73Δ leo1Δ strains transformed with empty vector, a low-copy plasmid expressing full-length HA-Rtf1, or high-copy plasmids expressing full-length Myc-Rtf1, wild type or mutant Myc-HMD63-152, or Myc-HMD74-139. An H2BK123R strain serves as a negative control. (C) Ribbon diagram of HMD74-139. Residues at which substitutions significantly diminished (yellow) or did not diminish (white) H2B K123ub are indicated. Dashes indicate hydrogen bonds. (D) Surface view of HMD74-139 with sequence identity among 73 fungal Rtf1 orthologs mapped onto the surface. Red indicates highest conservation. (E) Positions of residues that facilitate BPA-crosslinking to Rad6 (blue) cluster near E104 (magenta). Residues that failed to support Rad6 crosslinking are indicated in gray. (F) Residues that facilitate crosslinking to Rad6 interact with each other and to the other molecule of the HMD in the asymmetric unit. Molecule A (green) is shown with BPA crosslinking residues shown as blue sticks. E104 is colored magenta. Hydrogen bonding (yellow dashes) and van der Waals (blue dashes) interactions inferred from the structure are shown. See also Figures S2 and S4.

Article Snippet: Materials An antibody against human H2B K120ub (Cell Signaling #5546; 1:1000 dilution) was used to detect H2B K123ub in yeast ( Wozniak and Strahl, 2014 ).

Techniques: Western Blot, Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Negative Control, Sequencing

Journal: Molecular cell

Article Title: The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation Through an Interaction with Rad6

doi: 10.1016/j.molcel.2016.10.008

Figure Lengend Snippet: (A–E) Protein localization patterns as determined by ChIP-exo are centered at the +1 nucleosome dyad of coding genes. The gray fill, showing H2B occupancy as determined by ChIP-exo in a wild-type strain, is not to scale and should be used for positioning comparison only. The first three genic nucleosomes are indicated. Experiments were normalized using total tag count; thus the Y-axis represents an arbitrary linear scale for each separate trace and the absolute magnitudes are not comparable between different experiments. All experiments employed strains with epitope-tagged, endogenously expressed proteins (see Strain Table). (C) Rtf1, Rpb3, Spt4, and Spt5 localization at highly and lowly expressed genes (top and bottom quintiles), based on transcription frequency. (D–E) Occupancy of endogenously expressed Rad6 and Bre1 in strains containing or lacking RTF1 is shown for all genes (Panel D) or for genes in the highest and lowest expression quintiles (Panel E). See also Figure S6.

Article Snippet: Materials An antibody against human H2B K120ub (Cell Signaling #5546; 1:1000 dilution) was used to detect H2B K123ub in yeast ( Wozniak and Strahl, 2014 ).

Techniques: Expressing

Journal: Molecular cell

Article Title: The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation Through an Interaction with Rad6

doi: 10.1016/j.molcel.2016.10.008

Figure Lengend Snippet: (A–F) ChIP-exo data are presented as in Figure 4 and were normalized using total tag count unless otherwise indicated; thus the Y-axis represents an arbitrary linear scale for each separate trace and the absolute magnitudes are not comparable between different experiments. (A) Occupancy of H2B K123ub in a wild-type strain. H2B occupancy was determined in the same strain and is shown to scale. (B) Rtf1, Rpb3, Spt4, and Spt5 as in Figure 4B and 4C for genes with high and low H2B K123ub (top and bottom quintiles), based on H2B K123ub / bp values. (C) Occupancy of Rtf1, Rad6, and Bre1 for genes in the highest and lowest H2B K123ub quintiles. (D) Comparison of plasmid-expressed HSV-HMD63-152 and HSV-HMD63-152-E104K occupancy. To appreciate the difference in levels, datasets were normalized to the nucleosome free region. (E) Comparison of HA-Rtf1 and HA-Rtf1-E104K occupancy. Proteins were expressed from the endogenous RTF1 locus. (F) H2B K123ub localization in an rtf1Δ strain overexpressing either HSV-HMD63-152 (pHMD) or HSV-Rtf1 (pRtf1). H2B occupancy was determined in the same strains. Note that the H2B K123ub patterns are similar in strains containing a chromosomal copy of RTF1 (Figure 5A) or the high copy Rtf1 plasmid (pRtf1). (G) Box plot graphs of Rtf1 / HMD occupancy (left) and H2B K123ub / H2B ratio (right) in a wild-type strain, or in an rtf1Δ strain overexpressing either HSV-HMD63-152 or HSV-Rtf1 from a plasmid, for intergenic regions and transcription units of highly and lowly expressed genes. See also Figure S6.

Article Snippet: Materials An antibody against human H2B K120ub (Cell Signaling #5546; 1:1000 dilution) was used to detect H2B K123ub in yeast ( Wozniak and Strahl, 2014 ).

Techniques: Plasmid Preparation

Journal: Molecular cell

Article Title: The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation Through an Interaction with Rad6

doi: 10.1016/j.molcel.2016.10.008

Figure Lengend Snippet: (A and B) Unless otherwise indicated, in vitro ubiquitylation reactions contained: recombinant X. laevis nucleosomes, FLAG-yBre1 (E3), FLAG-hE1, His-HA-pK-Ubiquitin, and yRad6 (E2). HMD74-184, or an equivalent volume of storage buffer, was added to the reactions. Reactions were incubated in 1X Reaction Buffer at 30 °C for the indicated times and analyzed by Western blotting. H2A levels function as a loading control. Relative H2Bub was determined by setting the signal for lane 3 (Panel A) or lane 2 (Panel B) to one. (C) Top: Model showing relative enrichment of Pol II, Spt4-Spt5, Rad6-Bre1, Paf1C, and H2B K123ub along active genes in a wild-type background. Bottom: Model showing localization of the Rtf1 HMD and H2B K123ub in an rtf1Δ background when the HMD is expressed as the sole source of Rtf1 and cannot interact with Paf1C. The localization of Spt4-Spt5 and Rad6-Bre1 in the HMD-only strain has not been directly measured but is inferred from data in Figure 4D and previous work that showed Spt4 recruitment is unaffected in an rtf1Δ strain (Qiu et al., 2006). See also Figure S7.

Article Snippet: Materials An antibody against human H2B K120ub (Cell Signaling #5546; 1:1000 dilution) was used to detect H2B K123ub in yeast ( Wozniak and Strahl, 2014 ).

Techniques: In Vitro, Recombinant, Incubation, Western Blot