RIME analysis identifies ADNP as a co-regulatory partner of ZMYM2.

(A) Summary table of the ten top scoring interactors for ZMYM2. The average number of peptides across three RIME experiments are shown and whether detected previously in ZMYM2 IP-mass spectrometry experiments is indicated (1Yang et al., 2020; 2Connaughten et al., 2020). Core members of the ChAHP and GTF3C complexes are highlighted. (B) Depiction of interactions between ZMYM2 binding partners found in RIME experiments with known previous molecular interactions found in the STRING database (Jensen et al., 2009). (C) Co-immunoprecipitation analysis of ADNP with ZMYM2. Immunoprecipitation (IP) was performed with ZMYM2 or control IgG antibody from U2OS cells and resulting proteins detected by immunoblotting (IB) with the indicated antibodies. Molecular weight markers (kDa) and 10% input are shown. (D) De novo motif analysis of ADNP binding regions. The top five most significantly enriched motifs are shown, along with motif similarity to the indicated protein in brackets. (E) Venn diagram showing the overlap between ZMYM2 and ADNP binding regions. (F) UCSC genome browser of ChIP-seq data on example genomic loci showing binding of both ZMYM2 and ADNP. (G) Venn diagram showing overlaps in genes upregulated (left) or downregulated (right) following ADNP (orange) or ZMYM2 (red) depletion (fold change >1.6; Padj <0.01).

ZMYM2 interactions with molecularly distinct chromatin regions.

(A) Heatmaps showing the signals of the indicated proteins or chromatin marks from ChIP-seq experiments or the ATAC-seq signal in U2OS cells plotted across a 10 kb region surrounding the centres (arrowed) of the wild-type (WT) ZMYM2 binding regions. Clustering of the data produced 3 clusters. (B) Tag density plots of the indicated ChIP-seq or ATAC-seq signals in the three clusters of ZMYM2 binding regions. (C) UCSC genome browser of the indicated ChIP-seq and ATAC-seq data on example genomic loci from clusters 1 and 2.

Characterisation of ZMYM2 chromatin binding regions.

(A) De novo motif analysis of cluster 1 and 2 regions. The top five most significantly enriched motifs are shown with motif similarity to the indicated protein shown in brackets. (B) Heatmaps showing the signals of the indicated proteins or chromatin marks from ChIP-seq experiments in U2OS cells plotted across a 10 kb region surrounding the centres (arrowed) of the wild-type (WT) ZMYM2 binding regions. Clustering was retained from Fig. 2A and ADNP signal superimposed on top of this. The number and percentage of ZMYM2 peaks in each cluster overlapping with ADNP2 peaks is shown on the right. (C) Tag density plot of ADNP ChIP-seq signal from U2OS cells across a 10 kb region surrounding the centres (arrowed) of the three clusters of ZMYM2 binding regions. (D) Distribution of binding regions among different genomic categories for clusters 1 and 2 and the entire genome.

ZMYM2 interactions with TRIM28.

(A) Co-immunoprecipitation analysis of TRIM28 with ZMYM2. Immunoprecipitation (IP) was performed with ZMYM2 or control IgG antibody from U2OS cells and the resulting proteins detected by immunoblotting (IB) with the indicated antibodies. 10% input is shown (See Supplementary Fig. S3A for longer exposure). NEM was added to the extracts where indicated. (B) PLA assay of interactions between Myc-tagged ZMYM2 and endogenous TRIM28. Assays were carried out in U2OS cells transfected with a vector encoding Myc-ZMYM2 or left untransfected (UT). The addition of anti-Myc and -TRIM28 antibodies (ab) is indicated. Nuclei were stained with DAPI. Average numbers of foci per cell and numbers of cells are indicated (bottom). (C) Venn diagram showing the overlaps in downregulated (left) and upregulated (right) genes (Padj<0.05; fold change 2:1.6) from RNAseq analysis in U2OS cells treated with siRNAs against ZMYM2 or TRIM28. (D) Enriched GO terms of genes commonly upregulated and downregulated by ZMYM2 and TRIM28 depletion.

ZMYM2 location and gene expression.

(A) Numbers of differentially expressed genes following ZMYM2 depletion (left upregulated, right downregulated) whose TSS lies within the indicated distances of a ZMYM2 binding peak (black bars). Control sets of equal numbers of randomly selected genes are shown for comparison (grey bars showing the average of 10 datasets). P-values *= <0.05, **=<0.01, ***=<0.001. (B) Distance-dependent association of ZMYM2 binding regions with differentially regulated genes following depletion of ZMYM2 or TRIM28. The numbers in the boxes are the number of genes among the input gene sets (x-axis) that overlap with the ZMYM2 peaks (cluster 1 peaks only) at the indicated distances to TSS (y-axis) and the colour shows - log10 of p-value (Hypergeometric test). (C) Relative proportion of genes commonly up- or down-regulated following ZMYM2 or TRIM28 depletion in TADs which also contain ZMYM2 peaks from the indicated clusters or have no ZMYM2 peak in the same TADs. Significance relative no regions containing no peaks is shown P-value, ***=<0.001; ns= non-significant. (D) Boxplots of the relative distance of ZMYM2 binding regions from each of the clusters from TAD boundaries compared to two different control sets of randomly selected regions (n=2360). Statistical significance of cluster 1-3 distances compared to each of the control regions is shown (**= P-value <0.05 in all cases; student t-test).

ZMYM2 functionally associates with ERV repetitive elements.

(A) Percentage of the ZMYM2 binding sites in each of the indicated clusters containing each of the indicated classes of retrotransposon elements. The genome-wide proportion of genomic regions containing each type of the retrotransposon elements is also shown. (B) Proportions of LTR subclasses in cluster 1 and cluster 3 ZMYM2 binding regions. (C) UCSC genome browser view of a MER11A ERV1 LTR element located upstream of the HEATR6 locus, illustrating co-binding of ZMYM2 and TRIM28 (top) and a MIR SINE element located upstream of the EIF3D locus, illustrating co-binding of ZMYM2 and ADNP. (D) RT-qPCR analysis of expression of ZMYM2 and the indicated LTR elements following ZMYM2 depletion or control non-targeting (NT) siRNA treatment. Individual paired experiments are shown (n=4; P-values *=<0.05, ****=<0.0001). (E) Proportions of SINE subclasses in cluster 1-3 ZMYM2 binding regions. (F) RT-qPCR analysis of expression of ZMYM2 and the indicated SINE elements following ZMYM2 depletion or control non-targeting (NT) siRNA treatment. Individual paired experiments are shown (n=3; unpaired T-test P-values *= <0.05, **= <0.01, ****= <0.0001. (G) Model illustrating the two distinct complexes through which ZMYM2 functions on chromatin to control retrotransposon expression.