Supplementary Figure 4: Supporting analyses and experiments for the nature and function of 3D enhancer hubs (Related to Fig. 4).

a, Venn diagram showing overlap between previously assigned target genes for superenhancers (SE), newly identified SE target genes based on H3K27ac HiChIP contacts in PSCs, and genes connected to PSC-specific enhancer hubs, which represent enhancers contacting more than one gene according H3K27ac HiChIP (see also Fig. 4a). b, Comparison of the RNA levels of hub genes, non-hub genes or genes connected to SE in PSC samples as measured by RNA-seq and expressed as transcripts per million (TPM). All genes that are not connected to enhancer hubs, but are still detected within PSC-specific HiChIP loops were considered. Expression of all protein coding genes expressed in PSC ( > 1TPM) is shown as reference. Statistics were calculated by Wilcoxon rank sum test. n = 713 non-hub genes, n = 715 genes within hubs, n = 646 genes connected to SE and n = 22,000 total number of genes considered. c, RNA-seq signal (TPM) of Med13l -which is not part of the Tbx3 enhancer hub (see Fig. 4b)- during reprogramming. 2 biological replicates. d, Genotyping strategy and results confirming the homozygous deletion of the distal (left) or the proximal (right) Tbx3 enhancers. 2 KO PSC clones and 1 WT PSC clone. e, Example of an enhancer hub in PSCs. Normalized HiChIP signal around the viewpoint is illustrated as a virtual 4 C plot, showing average CPM across 2 biological replicates. Statistics were calculated with the R-package edgeR (see Methods for more details) f, H3K27ac ChIP-seq IGV tracks during reprogramming. g, Mean RNA-seq signal from 2 biological replicates of genes within the hub (Zic2 and Zic5), or nearby genes (Clybl and Pcca), are shown for each reprogramming stage to highlight concordance with H3K27ac HiChIP data and coordinated upregulation of genes within the hub. h, Schematic illustration of the CRSIPRi (dCas9-KRAB) targeting strategy for inactivation of the Zic2/Zic5 enhancer hub. i, RT-qPCR showing percentage expression of the enhancer RNA in dCas9-KRAB-targeted ESCs (n = 3 biological replicates) relative to matched WT samples (n = 3 biological replicates), normalized to an unaffected enhancer RNA (IG-DMR). P-values were calculated using paired one-tailed t-test. Error bar represent standard deviation and the measure of center is 9.63. j, RT-qPCR showing expression changes of genes within the hub (Zic2 and Zic5) and nearby genes (Clybl and Pcca) in dCas9-KRAB-targeted ESCs (n = 3 biological replicates), calculated as percentage relative to matched WT (n = 3 biological replicates) from three independent experiments after normalization to hprt expression. P-values were calculated using paired one-tailed t-test. Error bars represent standard deviation. For source data see Supplementary Table 11.