The H3K4me3 defect in Δ cells is independent of direct regulation of the Set1p complex

<p><b>Copyright information:</b></p><p>Taken from "Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex"</p><p></p><p>Nucleic Acids Research 2007;35(7):2428-2439.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874646.</p><p>© 2007 The Author(s)</p> () mRNA levels of Set1p complex components. RNA was extracted from exponentially growing BY4741 and Δ and subjected to northern blot analysis using the indicated probes. () Subunit composition of purified Set1p complexes. Strains containing or lacking 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 Δ 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 Δ cells. () 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) . Samples were separated on a 15% SDS-PAA gel, stained with Coomassie, dried and exposed to an X-ray film. () methylation of synthetic pre-methylated histone H3-tail peptides. Pre-methylated peptides were used as substrates for methylation using Set1p complexes purified from WT or Δ 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