Figure 3.
Changes in 5hmC Levels and Distribution at 5% Oxygen
(A) Global 5hmC levels as measured by mass spectrometry in two TSC lines (TS-GFP and TS-R26) in the stem cell state and upon differentiation for 3 days (Diff) at 20% and 5% O2 (n = 3 each). 5hmC levels are expressed as a percentage of 5hmC detected in undifferentiated TSCs at 20% O2. ∗p < 0.05, ∗∗p < 0.005 (unpaired t test).
(B) RNA-seq data showing differences in Tet1 expression levels in TSCs ("TS") at 20% and 5% O2, and TSCs differentiated for 3 days ("TS Diff") at 20% O2 and 5% O2 (n = 1 each).
(C) Global 5hmC levels, measured by mass spectrometry, in empty vector control TSCs and Tet1 KO TSCs generated by CRISPR (Chrysanthou et al., 2018) cultured at 20% and 5% O2 (n = 3 each). 5hmC levels are expressed as a percentage of the 5hmC amount detected in vector controls at 20% O2. ∗p < 0.05 (unpaired t test).
(D) Relative enrichment of 5hmC across different genomic features in TSCs grown at 20% (orange) and 5% O2 (light orange), and after 3 days differentiation at 20% (red) and 5% O2 (pink). hmeDIP-seq: n = 2 each. Enrichments were converted to a log2 scale.
(E) Box-whisker plot showing log2 normalized RNA-seq read counts over CGIs that lose 5hmC in TSCs differentiated for 3 days at 20% O2, as described in Figure 2. The box-whisker plot shows data from TSCs at 20% O2, and from TSCs differentiated for 3 days at 20% and 5% O2 (n = 1 each).
(F) Data store tree diagram based on a Pearson's correlation distance matrix of hmeDIP-seq reads mapping to CGIs in TSCs at 20% O2 and after 3 days differentiation in 20% and 5% O2.
(G) Heatmap of RNA-seq data showing expression levels (log2 RPKM) of TSC markers (Cdx2, Eomes, Elf5, Esrrb), giant cell markers (Tpbpa, prolactin-like genes) and syncytiotrophoblast markers (Gcm1, SynA, SynB) in TSCs at 20% O2, TSCs differentiated for 3 days at 20% O2, and TSCs differentiated for 3 days at 5% O2 (n = 1 each).
See also Figure S4.