Widespread reorganisation of pluripotent factor binding and gene regulatory interactions between human pluripotent states

The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.

The 'PIR' category describes the promoter-interacting regions. As expected, the most frequent type of interaction in both cell types was between baited protein-coding promoters and non-promoter regions.
c Hi-C and PCHi-C data (without significance filtering) were binned at a 25kb resolution and normalised   Genes coloured in red are differentially expressed between naive and primed PSCs (log10 fold change >1.5 or > -1.5 and with an adjusted P-value < 0.05). Other categories shown include genes that are not differentially expressed (log10 fold change <1.5 < -1.5 and/or with an adjusted P-value > 0.05) or genes that are not expressed (0 read counts in RNA-seq data).
The key landmarks of the networks are indicated (based on Fig. 1b) and are consistent in both network graphs, which provides support for the good reproducibility between replicate samples.  c Plot (left) shows the percentage of nodes within each community that are contained within the same TAD. For both cell types, the percentage was significantly higher compared to a set of randomly shuffled TAD coordinates. The inner box bounds the IQR divided by the median (horizontal line), and Spear-style whiskers extend to the minimum and maximum of the data values.
d Statistical test for (c). Bar chart shows the frequency of median percentages obtained after 500 random permutations of TAD coordinates (grey) and the comparison between this control set (Evperm) and the observed values (Evobs) using a permutation test (n=500) within the regioneR package (Gel et al. 2016).  Count of long-range interactions     c Heatmap shows the difference in HindIII fragment interaction frequency between cell types as a function of the chromatin state of the interacting regions (rows) and the linear interaction distance (columns, binned distances). Interacting regions that are engaged in long-range promoter interactions, defined as >1Mb, are highlighted by the dashed box. Nearly all (98%) of the long-range interactions were associated with bivalently-marked promoters and these regions have a higher interaction frequency in primed compared to naive PSCs.   (633) Genes interacting only in primed with a shared SE (250) Genes interacting in both naïve and primed with a shared SE (161) Genes interacting only in naïve with a naïve-specific SE (156) Genes interacting only in primed with a primed-specific SE (32)  e Genome browser representation shows an example of a naive-specific enhancer at the TBX3 locus.
TBX3 is highly expressed in naive PSCs, and the TBX3 promoter (shaded in red box) interacts with distal active enhancers (shaded in green boxes) only in naive PSCs. In primed PSCs, the TBX3 locus is decorated with H3K27me3, and interacts with distal genes that are also marked by H3K27me3, including LHX5 and TBX5.