Extended Data Fig. 10. Broad TET activity results in genome-wide 5hmC enrichment and methylation heterogeneity.

a) Mean 5hmC level in HUES64 differentiated motor neurons (MN) over gene bodies grouped into categories according to their expression status, d = day. The median is shown in bold, the box displays interquartile range and whiskers extend to 1.5x the interquartile range.

b) The difference (d60 – d16) in 5hmC vs. methylation for MNs at different genomic features. CGI = CpG island, TSS = transcription start site, IG = intergenic.

c) Transcript expression for genes involved in TET-mediated demethylation and base excision repair pathways.

d) For genomic features, the difference in methylation between ESCs is displayed.

e) Cell counts for HUES8 WT and TKO cells over 5d culture. Center points show the mean and error bars display the standard deviation for three independent cell culture replicates.

f) Schematic showing how stochastic turnover of cytosine modification can lead to intercellular heterogeneity. mC = methylcytosine (black circle), 5hmC = hydroxymethylcytosine (grey circle), C = unmethylated cytosine (white circle).

g) Representative browser tracks showing the methylation status for CpGs detected using hairpin bisulfite sequencing (BS; top) and WGBS (bottom). Each CpG covered by hairpin-BS is colored according to dyad methylation state.

h) Proportion of CpG dyads that were methylated, unmethylated or hemi-methylated in arrested HUES64 WT or PKO ESCs.

i) For regions enriched with different chromatin marks (ENCODE, H1 ESCs), the proportion of CpG dyads with each methylation status is displayed.

j) The percentage of CpG dyads that were hemi-methylated according to location within each genomic feature. TE = active typical enhancer.

k) Single cell RRBS data showing the percentage of unmethylated CpGs per cell. The violin plot extends from the data minima to the maxima where each dot is a single cell. Of CpGs that are normally methylated in bulk WGBS (≥ 0.9), we observed that 6.6% ±3.37 showed zero methylation in individual cells. These may have been targeted by TETs and are likely an underestimate as 5hmCs cannot be detected with our RRBS based approach.

l) The proportion of CpGs with zero, intermediate (not exactly zero or one) or one methylation status for arrested WT ESCs. In bulk populations, ESC arrest removes replication-associated heterogeneity and causes most CpGs to become fully methylated. However, 25% remain intermediate⁶⁶. The density plot shows methylation loss for each category of CpGs with intermediate CpGs showing the greatest loss suggesting they may be more frequently targeted by TETs hence TET activity may contribute to intermediate methylation levels in bulk populations.