Extended Data Fig. 3: Human HEK293 cells lacking MBD proteins show limited changes in transcription and global levels of DNA methylation.

a, Sequence of MBD loci in in HEK293 annotated as in Extended Data Fig. 1. b, Western blot for MBDs indicating absence of proteins. MBD3 protein levels unchanged (actin represents loading control). Blots representative of at least three independent experiments. c, Unsupervised clustering of RNA-seq (RPKM) samples from WT and MBD-QKO HEK293 cells treated with DMSO or 5-Aza-2′- deoxycytidine (Aza). d, Genes differentially expressed (FDR = < 0.01 and |log2FC| >= 1) are colored in blue or light green. Strongly upregulated genes in WT cells upon Aza treatment are colored in dark green (FDR = < 0.01 and log2FC > = 3). Comparing WT and MBD-QKO cells reveals that ~ 7-times more genes are upregulated upon Aza treatment, which tend to be transcriptionally silent in DMSO conditions. e, Hierarchical clustering of genes differentially expressed in MBD-QKO cells or in WT cells treated with Aza (blue or dark green points in d, n = 1265). Each row depicts the expression fold change (log2) of either: MBD-QKO vs. WT, WT treated with Aza vs. DMSO, MBD-QKO treated with Aza vs. DMSO. WT gene expression, WT promoter methylation and differentially expressed genes (DEG in black, FDR = < 0.01 and |log2FC| >= 1) are indicated. Of all genes that respond to Aza treatment in WT cells, only ~6 % (n = 20) of down- and 7 % (n = 110) of upregulated genes are also affected in MBD-QKO cells. The gene cluster with most upregulated genes under Aza treatment is marked with a red bar. These genes are largely unaffected after deletion of MBD proteins and are only de-repressed in MBD-QKO cells when treated with Aza. f, Gene ontology enrichment of genes represented in the cluster indicated by the red bar in e (Methods). g, Methylation frequencies in 1 kb windows comparing WT and MBD-QKO HEK293 cells (min. read coverage of 10 in both samples, n = ~2 mio). h, CpA methylation levels of 1 kb windows in HEK293 cells or human frontal cortex from Lister et. al.⁶⁶ (windows with min. read coverage of 100 in all samples). Boxplots as in Fig. 2d.