Figure 6.

Deletion of NOT4 diminishes H2B ubiquitylation and PAF complex recruitment. (A) NOT4 is required for efficient H2B ubiquitylation. Extracts of the indicated strains carrying a FLAG-HTB1 plasmid were separated on a 12.5% SDS-PAGE gel and subjected to western blot analysis using FLAG antibodies. The lower panel shows a shorter exposure of the same blot, indicating equal loading. (B) The ubiquitin-ligase activity of Not4p is not involved in the regulation of H3K4me3 levels. The H3K4 methylation status in extracts from cells expressing NOT4 or not4L35A alleles from the endogenous NOT4 locus and cells lacking NOT4 were subjected to western blot analysis. (C) Ctr9-HA is equally expressed in NOT4 and not4Δ cells. Strains expressing HA-tagged Ctr9p, containing or lacking NOT4, were used to determine Ctr9-HA levels by western blotting. H3K4me3 and TBP levels were used as controls. (D and E) NOT4 is required for PAF complex recruitment to the PYK1 and PGK1 ORFs. Strains from (C) were subjected to ChIP analysis of the PYK1 locus (amplicons are schematically depicted in upper panel). Exponentially growing cells were cross-linked and ChIPs were performed using anti-HA (12CA5) antibodies.