Extended Data Fig. 4: H3K4me3 loss does not have detectable effects on binding of TAF3, CDK7 and TBP to transcription start sites and to PIC formation.

(a) ChIP–seq profiles of the indicated proteins using DPY30–mAID and RBBP5–FKBP cells treated with or without Auxin/dTAG-13, respectively for 8 h. The enrichments were plotted over the transcription start sites (TSS ± 5 kb) of protein-coding genes. TSS, transcription start site. (b) Outline of the mass spectroscopy proteome profiling strategy for mapping the interaction networks of RNAPII in DPY30–mAID cells treated with or without Auxin. (c) String network of protein complexes (k-means clustering) showing RNAPII interactors in control cells compared with IgG mock IP. (d) Volcano plot showing proteins changing their association with RNAPII in response to acute loss of H3K4me3. The x axis displays the enrichment (log2 fold change) of proteins in Auxin-treated cells (Auxin 8 h) compared to DMSO-treated control cells (Auxin 0 h). The y axis shows the significance (-log10 P value) of enrichment calculated from three biological replicate experiments. A protein was considered an interactor if in one or both comparisons its levels were statistically significantly different (Q value ≤ 0.05, limma test, with P values adjusted by the Storey method). (e) RNAPII occupancy based on ChIP–seq in the indicated cell lines. Metagene analysis showing the genome-wide enrichment averages on protein-coding genes, data are shown along with 3 kb upstream of the transcriptional start site to 5 kb downstream of the end of each annotated gene. TSS, transcription start site, TES, transcription end site. (f) Estimation of a gene’s “pausing index” (PI) from RNAPII ChIP–seq data. The promoter is defined as the region covering 200 bp upstream to 200 bp downstream of the TSS; the gene body is defined as the region from 400 bp downstream of the TSS to TES, genes with gene length less than 400 bp are removed for pausing index analysis. (g) Violin plots showing changes of gene expression in the indicated samples. Genes were separated into three equal parts based on their accumulation change of RNAPII in promoter-proximal region. (h) An IGV snapshot comparing RNAPII, RNAPII Ser 2p and Ser 5p ChIP–seq signals in control and Auxin-treated DPY30–mAID cells at the indicated times. (i) Average metagene ChIP–seq profiles for the indicated factors in control and dTAG-13-treated RBBP5–FKBP cells.