Specific requirement for the NOT genes for global H3K4 tri-methylation. (A) Histone H3K4 mono-, di- and tri-methylation levels were determined in lysates from logarithmically growing BY4741-based CCR4-NOT gene deletion strains. Protein extracts of the indicated strains were separated by SDS-PAGE (15%) and subjected to western blotting using H3K4me1-, H3K4me2- and H3K4me3- specific antibodies. TBP levels were determined as a loading control. (B) MY1-based NOT mutant strains were used to determine H3K4me2, H3K4me3 and total H3 levels as in (A). H3K4me3 and H3K4me2 levels were quantified using ImageQuant software and represented as relative to WT after normalization to total H3 levels. (C) Direct comparison of H3K4me2 and H3K4me3 levels in BY4741, not4Δ, not5Δ and spp1Δ strains. Analysis was performed as in (A). (D) H3K79me2 and H3K79me3 levels in the indicated BY4741 deletion strains. Analysis was performed as in (A). (E) Silencing assay using strains, containing URA3 in the telomeric region of the left arm of chromosome 7, lacking NOT4 or DOT1. Cells were spotted in 5-fold serial dilutions on SC plates or on SC plates containing 5FOA.

Decreased H3K4me3 in not4Δ cells is not necessarily due to decreased RNA polymerase II loading (A) ChIP analysis of H3K4me3 and RNA pol II levels on the HSP104 locus. Data are represented as relative to WT. ChIP efficiencies were 2–3% and 0.15–0.2% for H3K4me3 and 8WG16 antibodies, respectively. (B) Deletion of NOT4 or SPP1 lead to a similar decrease in H3K4me3 and transcript levels on the PYK1 gene. Chromatin extracts of BY4741, not4Δ and spp1Δ strains were subjected to ChIP analysis using H3K4me3-specific antibodies. PYK1 mRNA transcript levels are affected by deletion of NOT4 or SPP1. PYK1 mRNA in total RNA of BY4147, not4Δ and spp1Δ strains was analyzed by quantitative reverse-transcriptase PCR.

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.

A model indicating how the Ccr4-Not complex may function in establishing H3K4 tri-methylation through regulation of PAF complex recruitment. The Ccr4-Not complex could be controlled by the Bur1/2 kinase to facilitate PAF complex recruitment (downstream). Alternatively, the Bur1/2 and Ccr4-Not complexes function in distinct pathways sharing several components and independently regulate PAF complex recruitment (parallel).

Ccr4-Not complex components genetically interact with BUR1 and BUR2 and play a role in transcription elongation. (A) Tetrad analysis of bur2Δ (KMY201) and BY4741 Ccr4-Not deletion or not4Δ (KMY40) BY4741bur2Δ diploids. Circles indicate double knock-out strains. Relevant genotypes of the haploid strains obtained are indicated (B) Plasmid-shuffle analysis of bur1-23 and not4Δ double-mutant strains containing pRS316-BUR1. Here, 10-fold serial dilutions of the indicated strains were spotted on SC and SC containing 0.1% 5FOA. (C) 6-Azauracil sensitivity assay. Here, 10-fold serial dilutions of the indicated strains were spotted on SC-U and SC-U containing 6-AU (100 μg/ml). (D) In vivo transcription elongation analysis of a long GC-rich reporter. BY4741 and not4Δ (KMY58) strains transformed with a GAL1pr-LacZ plasmid (pGR422) were grown in a medium containing raffinose and shifted to a medium containing galactose for 0, 45 and 90 min. Northern blot analysis on LacZ and endogenous GAL1 mRNA was performed using 18S rRNA as a control.

NOT4 is required for H3K4 tri-methylation but not di-methylation of the PYK1 ORF. (A) Schematic representation of the PYK1 locus and the amplicons used in (B–H). (B) ChIP analysis of the PYK1 ORF in BY4741 and not4Δ strains. Exponentially growing cells were subjected to ChIP analysis using H3K4me3 antibodies. (C) As in (A), using H3K4me2 antibodies. (D) As in (A), using H3K36me2 antibodies. (E) As in (A), using H3K79me2 antibodies. (F) As in (A), using CTD antibodies (8WG16). (G) Quantitative reverse-transcriptase PCR analysis of PYK1 and TUB1 mRNA levels in BY4741 WT and not4Δ strains. (H) ChIP analysis of H3K4me3 levels in MY1 WT and not2Δ cells. (I) ChIP analysis of H3K4me2 levels in MY1 WT and not2Δ cells. (J) Not4p is recruited to the 5′ region of the PYK1 ORF. Strains expressing mycAVI-tagged Not4p were transformed with a BirA expression plasmid and subjected to ChIP analysis. Signals were normalized to an empty plasmid control.

The H3K4me3 defect in not4Δ cells is independent of direct regulation of the Set1p complex. (A) mRNA levels of Set1p complex components. RNA was extracted from exponentially growing BY4741 and not4Δ and subjected to northern blot analysis using the indicated probes. (B) Subunit composition of purified Set1p complexes. Strains containing or lacking NOT4 and expressing a TAP-tagged Bre2p were used to purify Set1p complexes (upper panel). Equal expression of Bre2-TAP proteins was checked by western blot analysis (lower panel). The indicated proteins were identified using LC-MS/MS. Several additional proteins were present in the not4Δ purification when compared to the WT. Mass spectrometry showed that these bands contained non-related lysosomal proteins. We consider this an artifact of the purification, likely caused by differences in the concentration of the lysates of the WT and not4Δ cells. (C) Histone methyl transferase assay using purified Set1p complexes. Increasing amounts of Set1p complex or a mock-purification control were use to methylate soluble histones (from calf thymus) in vitro. Samples were separated on a 15% SDS-PAA gel, stained with Coomassie, dried and exposed to an X-ray film. (D) In vitro methylation of synthetic pre-methylated histone H3-tail peptides. Pre-methylated peptides were used as substrates for in vitro methylation using Set1p complexes purified from WT or not4Δ cells. Samples were separated on a 20% SDS-PAA gel, dried and exposed to an X-ray film. Signals were quantified using ImageQuant software and represented as arbitrary units after background correction.

The Ccr4-Not complex functions downstream of or parallel to the Bur1/2 kinase. (A) Bur1-HA and Bur2-HA are equally expressed in WT and not4Δ cells. Extracts of cells expressing HA-tagged Bur1p or Bur2p containing or lacking NOT4, or a non-tagged control, were subjected to western blot analysis using antibodies against the HA-tag, H3K4me3 and TBP. (B) Recruitment of the Bur1/2 complex to the PYK1 locus does not depend on NOT4. WT and not4Δ cells expressing HA-tagged Bur1p or Bur2p, and a no-tag control, were subjected to ChIP analysis using anti-HA (12CA5) antibodies (amplicons are schematically depicted in the upper panel). (C) As in (B), except that the analysis was performed for the PGK1 locus.