Sequential deregulation of histone marks, chromatin accessibility and gene expression in response to PROTAC-induced degradation of ASH2L

The trithorax protein ASH2L is essential for organismal and tissue development. As a subunit of COMPASS/KMT2 complexes, ASH2L is necessary for methylation of histone H3 lysine 4 (H3K4). Mono- and tri-methylation at this site mark active enhancers and promoters, respectively, although the functional relevance of H3K4 methylation is only partially understood. ASH2L has a long half-life, which results in a slow decrease upon knockout. This has made it difficult to define direct consequences. To overcome this limitation, we employed a PROTAC system to rapidly degrade ASH2L and address direct effects. ASH2L loss resulted in inhibition of proliferation of mouse embryo fibroblasts. Shortly after ASH2L degradation H3K4me3 decreased with its half-life varying between promoters. Subsequently, H3K4me1 increased at promoters and decreased at some enhancers. H3K27ac and H3K27me3, histone marks closely linked to H3K4 methylation, were affected with considerable delay. In parallel, chromatin compaction increased at promoters. Of note, nascent gene transcription was not affected early but overall RNA expression was deregulated late after ASH2L loss. Together, these findings suggest that downstream effects are ordered but relatively slow, despite the rapid loss of ASH2L and inactivation of KMT2 complexes. It appears that the systems that control gene transcription are well buffered and strong effects are only beginning to unfold after considerable delay.

Supplementary Figure S1 (supporting Figures 1 and 2  (e) Down-regulated genes, which encode cell cycle regulators and replication factors after dTAG-13 treatment for 24 hrs (from Supplementary Table S1).

Supplementary Tables
All sequencing data are available in NCBI's Gene Expression Omnibus as SuperSeries under accession number GSE241174: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241174.Table S1 List of significantly changed genes after 24h compared to the WT (RNA-seq); available in GEO under accession number GSE240987 Table S2a All identified Ash2l peaks across all the samples; available in GEO under accession number GSE24100.1Table S2b List of significantly changed Ash2l binding sites after 1h compared to the WT (ChIP-seq Ash2l) ; available in GEO under accession number GSE241001.Table S3a All identified H3K4me3 peaks across all the samples; available in GEO under accession number GSE240994.Table S3b List of significantly changed H3K4me3 binding sites in each timepoint compared to the WT (ChIP-seq H3K4me3); available in GEO under accession number GSE240994.Table S4a All identified H3K4me1 peaks across all the samples; available in GEO under accession number GSE240992.Table S4b List of significantly changed H3K4me1 binding sites in each timepoint compared to the WT (ChIP-seq H3K4me1); available in GEO under accession number GSE240992.Table S5a All identified H3K27ac peaks across all the samples; available in GEO under accession number GSE240990.Table S5b List of significantly changed H3K27ac binding sites in each timepoint compared to the WT (ChIP-seq H3K27ac); available in GEO under accession number GSE240990.Table S6a All identified H3K27me3 peaks across all the samples (ChIP-seq H3K27me3_First experiment); available in GEO under accession number GSE240999.Table S6b List of significantly changed H3K27me3 binding sites in each timepoint compared to the WT (ChIP-seq H3K27me3_First experiment); available in GEO under accession number GSE240999.Table S7a All identified H3K27me3 peaks across all the samples (ChIP-seq H3K27me3_Second experiment); available in GEO under accession number GSE241000.Table S7b List of significantly changed H3K27me3 binding sites in each timepoint compared to the WT (ChIP-seq H3K27me3_Second experiment); available in GEO under accession number GSE241000.Table S8a All identified peaks across all the samples (ATAC-seq); available in GEO under accession number GSE241169.Table S8b List of sites significantly changed in accessibility in each timepoint compared to the WT (ATAC-seq); available in GEO under accession number GSE241169.Table S9 List of all identified putative Enhancers; available in GEO under accession number GSE240994.Table S10 List of significantly changed TF (ATAC-seq; differential analysis using RGT-Hint); available in GEO under accession number GSE241169.Table S11 List of significantly changed genes after 4h or 8h compared to the WT (Nascent RNA; Click-it); available in GEO under accession number GSE239789.
) (a) Schematic summary of the steps employed to generate the mouse embryo fibroblast clones that are dependent on the FKBP-HA2-ASH2L fusion protein.The CAGG-Cre-ER transgene contains the CMV-IE enhancer/chicken β-actin/rabbit β-globin hybrid promoter and encodes for a CreER fusion protein{Hayashi, 2002 #1151}.(b) RT-qPCR analysis of exon 4 of Ash2l from iMEF cells treated ±hydroxytamoxifen (-/+) or from the NG3 and ND10 cell clones.(c-e) FKBP-HA2-ASH2L expressing cells were treated with dTAG-13.The indicated proteins were identified on Western blots, the cell clones analyzed are indicated.
(f) Analysis of the WRAD components Wdr5 and Rbbp5 upon loss of FKBP-HA2-ASH2L.dTAG-13 (100 nM) treatment was for the times indicated.Tubulin is shown for control.Supplementary Figure S3 (supporting Figure 3) (a) Cells were treated with or without dTAG-13 (100 nM) for 1 or 16 hrs.ChIP-qPCR were performed using either HA-specific antibodies to immunoprecipitate FKBP-HA2-ASH2L or control antibodies.Mean values of two replicates are displayed.(b) Heatmaps showing sample-to-sample distances comparing all ChIP-seq samples of the indicated histone marks for both biological replicates to each other.(c)Biological pathways associated with the genes associated with the 663 downregulated H3K4me3 sites after 2 hours upon loss of ASH2L (GO "biological process", q value <0.05).
ChIP-qPCR analyses of selected promoters for the indicated histone marks.Indicated are mean values ±SD of 4 to 5 measurements.(* p≤0.05; *** ≤ 0.001; **** p ≤ 0.0001) (e) Screen shots of integrative genomics viewer (IGV) of the Egr1 promoter region showing the normalized BigWig tracks (normalized using counts per Million) of ChIP-seq experiments of the indicated histone marks and of ASH2L.Additionally, the chromatin accessibility is summarized as measured by ATAC-seq at the indicated timepoints after dTAG-13 treatment.Supplementary FigureS4(supporting Figure4) (a) H3K4me3 binding sites (25431) were divided into three equal groups (high, medium and low, 8477 each).A window of ±1 kb around transcriptional start sites of all promoters was considered.The total number of binding sites within this window at promoters in the categories high, medium and low is 7937, 5413 and 1955, respectively.The log2 fold changes of signals of the indicated histone marks at the promoters of these three classes are displayed.For H3K27me3 the 2, 4 and 8 hour time points are not shown as no changes were observed.(b) Gene expression as determined by RNA-seq after 24 hours of dTAG-13 treatment was linked to promoters with high, medium and low H3K4me3 in control cells.
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