KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks

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Abstract

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer–promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.

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Fig. 1: Dynamic KLF4 binding during reprogramming and association with chromatin accessibility and enhancer activity.
Fig. 2: Characterization of the 3D enhancer connectomes in MEFs and PSCs by H3K27ac HiChIP analysis.
Fig. 3: Highly connected enhancers are characterized by specific features.
Fig. 4: Coregulation of genes within highly interacting enhancer hubs.
Fig. 5: Chromatin reorganization around KLF4 binding sites during reprogramming associates with enhancer rewiring and requires additional cofactors.
Fig. 6: Inducible depletion of KLF proteins induces 3D enhancer reorganization and concordant transcriptional changes.
Fig. 7: Disruption of the KLF4 binding site within Tbx3 and Zfp42 enhancers induces looping abrogation and downregulation of linked genes in PSCs.

Data availability

All genomics data (RNA-seq, ChIP-seq, ATAC-seq and HiChIP) have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE113431. RIME data have deposited to the ProteomeXchange Consortium via the PRIDE partner repository under the identifier PXD014631 (www.ebi.ac.uk/pride). The accession codes for all previously published datasets that were used in this study at listed in Supplementary Table 12. The source data for Figs. 4f, 6f,g, 7d,e,i,j and Supplementary Figs. 4i,j, 6b, 7d have been provided as Supplementary Table 11.

Code availability

The computational code for the processing of HiChIP/HiC is available under https://github.com/NYU-BFX/hic-bench. All other custom computational code are available from the corresponding author on request.

Change history

  • 07 October 2019

    In the HTML version of this article originally published, the contact information for co-corresponding author Aristotelis Tsirigos was not included. This should be linked in the author list, as well as in the correspondence section, which should read ‘Corresponding authors – Aristotelis Tsirigos or Effie Apostolou’. This has now been corrected.

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Acknowledgements

We are grateful to A. Melnick and the members of the Apostolou, Tsirigos and Stadtfeld laboratories for critical reading of the manuscript. WWe applied an unpaired, one-sidede also want to thank Z. Chen and the Biostatistics and Epidemiology Consulting Service for the advice and final evaluation on the statistical tools and analyses and L. Dow for sharing the CRISPR–Cas9 vectors. D.C.G. was supported by the New York Stem Cell Foundation and the Family-Friendly Postdoctoral Initiative at Weill Cornell Medicine. A.A. is supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. A.T. is supported by the American Cancer Society (grant no. RSG-15-189-01-RMC), the Leukemia and Lymphoma Society and the St. Baldrick’s Foundation. E.A. is supported by the NIH Director’s New Innovator Award (grant no. DP2DA043813) and the Tri-Institutional Stem Cell Initiative by the Starr Foundation.

Author information

E.A. conceived, designed and supervised the study, and wrote the manuscript together with D.C.D.G., with help from all of the authors. D.C.D.G. performed all of the experiments with help from D.K. and V.S. A.K. and A.P. performed all HiChIP, HiC and integrative computational analyses under the guidance of A.T. Y.L. performed the initial ChIP-seq, RNA-seq and ATAC-seq analyses. D.M. performed the HiC and CRISPRi experiments using a stable dCas9–KRAB ESC line generated by B.A. A.A. performed the RIME experiments and iPSC ChIP-seq. P.C. and N.D. ran and analysed the RIME results. M.S. provided the reprogrammable cells and guidance on the reprogramming experiments.

Correspondence to Aristotelis Tsirigos or Effie Apostolou.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Isolation and molecular characterization of reprogramming intermediates (related to Fig. 1).

a, FACS analysis plots showing expression of SSEA1 (early pluripotency marker) and Thy1 (somatic marker) at different stages of reprogramming, before and after SSEA1 enrichment by MACS isolation. Representative plots from 2 biological replicates. b, Pie charts of functional classification of KLF4 Early, Mid, Late and Transient peaks (based on chromatin states8) (piPSC = partial iPSCs). c, PCA analysis of ATAC-seq peaks (108373 accessible sites) in MEF, PSC and different stages of reprogramming. d, Average line plot showing the methylated CG to non-methylated CG ratio from MEF data12 centred (+/-2.5Kb) around different clusters of KLF4 binding sites (Early (n = 6275), Mid (n = 3712), Late (n = 9287) and Transient (n = 17891) KLF4 targets, Fig. 2b). e, Motif enrichment for Early, Mid, Late and Transient KLF4 binding sites. Selected factors are shown and their significance is expressed as Z-score of –log10(pvalue) (hypegeometric test) (left) or z-score of motif frequency (right). f, PCA analysis of H3K27ac ChIP-seq peaks (106751 H3K27ac peaks) called in MEF, PSC and different stages of reprogramming g, PCA of RNA-seq (22 K genes) in MEF, PSC and different stages of reprogramming. h, Line plots of the median expression (red line) of genes closest to Early, Mid, Late and Transient peaks, expressed as TPM (transcripts per million) with their corresponding confidence intervals (CI) calculated as described in the Methods.

Supplementary Figure 2 HiChIP and HiC pipelines and comparisons (related to Fig. 2).

a, Schematic work-flow for HiChIP and HiC analysis. b, Percentages of PSC-specific, constant or MEF-specific H3K27ac HiChIP loops that were detected in HiC experiments (either generated in-house or published ultra-resolution HiC in PSC38). c, Normalized HiChiP (top) and HiC (bottom) signals in MEF and PSC are illustrated in a virtual 4 C format around the indicated viewpoint (Tbx3 promoter). Signal is shown as average CPM across 2 biological replicates. Representative H3K27ac ChIP-seq tracks are shown in MEF and PSC from 2 biological replicates d, Violin plot representing log2 fold change of distance-normalized HiC signal in PSCs versus MEFs of MEF-specific, constant and PSC-specific loops as called by H3K27ac HiChIP. Only contacts detected as significant in HiC data were considered. Numbers of considered loops per category are shown in parenthesis n = 9747 for MEF-specific, n = 5681 for constant and n = 8975 for PSC-specific. [Unpaired two-tailed t-test was used to determine the p-value. The minimum, maximum values and q25%, q50% and q75% are shown in each violin.

Supplementary Figure 3 HiChIP and HiC connectivity (related to Fig. 3).

a, Histogram of anchor connectivity based on H3K27ac MEF and PSC HiChIP called loops. The numbers of contacts per anchor are grouped as shown in the bottom and the actual number of anchors is depicted on top of each bar. b, Connectivity of MEF or PSC anchors based on HiC-called loops represented as number of high-confidence contacts around each 10 kb anchor. Statistics were calculated by Wilcoxon rank sum test. (min = 1, max = 27, q25% = 1, q50% = 3, q75% = 6; n = 115900 unique MEF anchors : min = 1, max = 36, q25% = 2, q50% = 5, q75% = 8; n = 111213 unique PSC anchors). c, Scatter plot showing the correlation (r = 0.289) of H3K27ac ChIP-seq strength (sum of H3K27ac ChIP/input of all peaks within the anchor) with the number of H3K27ac HiChIP contacts per anchor in PSCs.

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.

Supplementary Figure 5 QC for KLF4 HiChIP and supporting evidence for the distinct categories of KLF4 HiChIP contacts (Related to Fig. 5).

a, PCA analysis of loops called as significant by H3K27ac and KLF4 HiChIP in different samples. b, Left: Chromatin loops that were detected by both KLF4 and H3k27ac HiChIP in PSCs were clustered based on the timing of KLF4 binding and looping during reprogramming. Right: Line plot showing expression changes of genes that belong to each of the indicated loop categories during reprogramming (median values are plotted relative to PSC). c, Pie chart showing the percentage of KLF4 PSC loops that were also detected by H3K27ac HiChIP in PSCs (H3K27ac-dependent) or not (H3K27ac-independent). d, Boxplot showing the significant difference (two-sided Wilcoxon rank sum test) between the expression of genes within all anchors of KLF4-mediated loops that are either H3K27ac-dependent. Boxplots are showing the minimum, maximum and q25%, q50% and q75% values. Number of genes considered for the statistical test is n = 5824 genes within H3K27ac-dependent and n = 2472 genes within H3K27ac-independent KLF4 HiChIP loops. e, Gene ontology for genes within anchors of H3K27ac-dependent (n = 5824) or -independent (n = 2472) KLF4 loops. Enrichment and significance of GO terms was calculated with DAVID knowledgebase and two-sided Fisher’s exact test. f, Proposed model for different categories of chromatin loops mediated by KLF4 and cofactors. Example genes are reported for each category.

Supplementary Figure 6 Supporting results for the inducible TKO PSC line and connectivity analyses (Related to Fig. 6).

a, Western blot analysis showing KLF4 protein levels before (0) and after (48 hr) dox induction in 2 biological replicates ESC clones that harbour dox-inducible CRISPR–Cas9 and gRNAs that target the Klf4 gene (KLF4 KO1 and KLF4 KO2). Relative position and size (kDa) of protein marker is shown on the right. (See also Supplementary Fig. 8). b, RT-qPCR showing elevated levels of Klf2 and Klf5 genes in dox-induced KLF4 KO ESCs. Paired one-tailed Student’s t-test was used to determine significance. n = 2 biological replicates c, Representative Western blot analysis from n = 2 independent experiments showing levels of indicated proteins in a clonal population of ESCs containing an inducible CRISPR–Cas9 construct and gRNAs that target the Klf2, Klf5 and Klf4 genes. Cells were either untreated (0, wild type or WT cells) or treated with dox for 24 hours (triple knock-out or TKO). Relative position and size (kDa) of protein marker is shown on the right. (See also Supplementary Fig. 8). d, Boxplot showing the sum connectivity of all H3K27ac HiChIP anchors in WT or TKO ESCs (WT/TKO) within each enhancer category (n = 348 for hubs, n = 231 for SE and n = 8563 for TE). Significance was calculated using two-sided Wilcoxon rank sum test. The minimum, maximum and q25%, q50% and q75% values are shown.

Supplementary Figure 7 Supporting results for the genetic targeting of KLF4 binding site within the Tbx3 hub (Related to Figure 7).

a, IGV tracks of H3K27ac and KLF4 ChIP-seq in PSCs showing the whole Tbx3 distal enhancer (top), the region that was deleted by CRISPR/Cas9 (Dist-KO, bottom, see Fig. 4f) and the location of the gRNA used to mutate a specific KLF4 binding motif (Dis-KLF4mut gRNA). b, Genotyping strategy of the surveyor assay used to detect mutation/indel at the target KLF4 binding site within the distal Tbx3 enhancer (Dis-KLF4mut). The results for 4 homozygous mutant clones (mut1-4) are shown (See also Supplementary Fig. 8). c, Sequencing results of the four mutant (mut) clones compared to the wild type (WT). d, ChIP-qPCR showing the relative levels of KLF4 binding to Tbx3 distal enhancer in n = 2 WT clones and n = 4 mut clones (left panel). Values show ChIP signal over input. As control, binding of KLF4 to an unaffected region (Fbxo15 promoter) was tested (right panel). Unpaired on-tailed t-test was performed to compare WT vs MUT and the specific p-values are shown in the graph.

Supplementary Figure 8

Unprocessed images of all gels and blots.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Tables titles/legends

Reporting Summary

Supplementary Table 1

KLF4 ChIP-seq peaks during reprogramming.

Supplementary Table 2

H3K27ac ChIP-seq peaks during reprogramming.

Supplementary Table 3

TPM values for RNA-seq in MEF, PSC and different stages of reprogramming.

Supplementary Table 4

Coordinates of H3K27ac HiChIP MEF-specific loops, PSC-specific loops and constant.

Supplementary Table 5

Coordinates of enhancer hubs.

Supplementary Table 6

Coordinates of KLF4 HiChIP gained loops and lost loops.

Supplementary Table 7

List of proteins found to interact with KLF4 by RIME.

Supplementary Table 8

Coordinates of H3K27ac HiChIP in WT and TKO cells.

Supplementary Table 9

Primers and gRNAs used in this study.

Supplementary Table 10

Antibody information.

Supplementary Table 11

Statistics source data.

Supplementary Table 12

Accession codes of published datasets used in this study.

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Di Giammartino, D.C., Kloetgen, A., Polyzos, A. et al. KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks. Nat Cell Biol 21, 1179–1190 (2019) doi:10.1038/s41556-019-0390-6

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