KLF4 fused to mTurquoise2 (KLF4-mTurq) localizes to the nucleus and forms small puncta or round droplets, whereas mTurquoise2 alone is diffusely distributed throughout the nucleus and the cytoplasm (Fig. We used expression tags to monitor the distribution of KLF4 by fluorescence microscopy in HEK 293T cells or BJ fibroblasts, the somatic cells most widely used for reprogramming 28. KLF4 forms nuclear condensates at modest expression levels We propose that bridging and/or condensation with DNA in a sequence- and CpG methylation-dependent manner underlie KLF4 function as a key chromatin organizer and pioneer transcription factor in somatic cell reprogramming. Single molecule methods show that KLF4 tandem zinc fingers bring together short DNA duplexes in dilute solution by a bridging interaction. KLF4 DBD condensation with a NANOG promoter duplex is strongly enhanced by CpG methylation of a KLF4 cognate site, and ZnF point mutations that weaken interactions with DNA cognate sites decrease condensation in cells and in vitro. Surprisingly, the intrinsically disordered region is not essential for KLF4 condensation in cells, and a KLF4 fragment comprising the isolated DNA binding domain (DBD) condenses with DNA in vitro. Here, we show that KLF4 forms a liquid-like biomolecular condensate with DNA that recruits OCT4 and SOX2. The ability of KLF4 to undergo biomolecular condensation could facilitate pioneer interactions with closed chromatin and, as others have speculated 15, might stabilize long-range contacts between genomic loci. The KLF4 DNA binding domain and IDR might participate in such processes in open chromatin, and the KLF4 preference for CpG methylated over unmethylated cognate sites 27 combined with its ability to bind 6 bp partial sites in nucleosomal DNA 16 could help target it to silenced chromatin.
In current models for transcriptional activation, TFs bound to their cognate sites cooperate with co-localized co-activators to recruit Mediator complex and RNA polymerase II through IDR:IDR mediated biomolecular condensations 23, 24, 25, 26. The first 400 KLF4 residues are likely to be disordered because they have low sequence complexity, and intrinsically disordered regions (IDRs) of other TFs help to silence 21, 22 or activate 23, 24, 25, 26 gene expression. KLF4 contacts the DNA major groove with three tandem C 2H 2 zinc fingers (ZnFs) that make specific interactions 17, 18 at 9 base pair (bp) cognate DNA sites 19, 20. How KLF4 or other TFs might initiate chromatin reorganizations that determine cell fate is of intense interest 3, 15, 16. KLF4 is enriched at ESC super-enhancers 14 and at iPSC genomic anchors that make more than four contacts, further implicating KLF4 in chromatin organization 15. In PSCs, KLF4 is enriched at the NANOG 12 and OCT4 13 loci, which interact through space with many other pluripotency-related genomic sites. KLF4 cooperates with transcription factors (TFs) OCT4 and SOX2 in reprogramming to silence somatic enhancers and activate enhancers of pluripotency genes 5, 6, including the ‘gateway to pluripotency’ gene NANOG 7, which is highly expressed in embryonic stem cell (ESCs) 8, 9, 10, 11.
Krüppel like factor 4 (KLF4) is a key constituent of reprogramming cocktails that transform fibroblasts to induced pluripotent stem cells (iPSCs) 1, 2, 3, 4. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state. Liquid–liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region.
Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency.