Dissecting OCT4 defines the role of nucleosome binding in pluripotency

Abstract

Pioneer transcription factors (TFs) such as OCT4 can target silent genes embedded in nucleosome-dense regions. How nucleosome interaction enables TFs to target chromatin and determine cell identity remains elusive. Here, we systematically dissect OCT4 to show that nucleosome binding is encoded within the DNA-binding domain and yet can be uncoupled from free DNA binding. Furthermore, accelerating the binding kinetics of OCT4 to DNA enhances nucleosome binding. In cells, uncoupling nucleosome binding diminishes the ability of OCT4 to individually access closed chromatin, while more dynamic nucleosome binding results in expansive genome scanning within closed chromatin. However, both uncoupling and enhancing nucleosome binding are detrimental to inducing pluripotency from differentiated cells. Remarkably, stable interactions between OCT4 and nucleosomes are continuously required for maintaining the accessibility of pluripotency enhancers in stem cells. Our findings reveal how the affinity and residence time of OCT4-nucleosome complexes modulate chromatin accessibility during cell fate changes and maintenance. 3 MAIN To maintain cell identity, TFs are often associated with accessible enhancers and promoters of active genes. However, during cell fate changes, certain TFs target silent genes within closed chromatin, acting as "pioneer factors" 1 . For instance, Oct4, Sox2, Klf4 and c-Myc (OSKM) predominantly occupy open chromatin to maintain pluripotency in embryonic stem cells (ESCs) 2-4 . Yet, during early reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs), OSK, but not c-Myc, act as pioneer factors 5-9 .Nonetheless, binding of OSK to open chromatin has also been suggested to be important for reprogramming 10,11 . To date, it has not been possible to separate the conventional open chromatin binding from closed chromatin targeting, limiting the ability to directly examine the role of pioneer activity in pluripotency.We have previously shown that OSK can directly interact with nucleosomes like the paradigm pioneer factor FoxA, supporting a link between pioneer activity and nucleosome binding [12][13][14][15][16][17] . A systematic evolution of ligands by exponential enrichment (SELEX) study has revealed that a wide variety of DNA-binding domains (DBDs) can bind nucleosomes in vitro 18 . Commonly, DBDs containing short anchoring α helices have been shown to interact most strongly with nucleosomes in vitro 19 . However, FoxA-DBD requires an extra helical region to bind nucleosomes and open chromatin, suggesting that DBDs may not contain the full pioneer capacity 15,20 . Recently, the cryo-EM structure of OCT4-DBD co-bound with SOX2-DBD to an engineered nucleosome has shown a potential OCT4-SOX2 nucleosome readout that involves the OCT4 POU-specific domain (POUS) and SOX2 high mobility group (HMG), but not the POU-homeodomain (POUHD) of OCT4-DBD 21 . The cooperative interaction between OCT4 and SOX2 is critical for pluripotency

B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening mediated by pioneering factors

“…Cluster 2 indicates that removal of ZFP266 facilitates OSKM-mediated chromatin opening. In fact, we observed OSK binding in cluster 2 reprogramming MORs with particularly strong KLF4 signal using published reprogramming 48-hour ChIP-Seq datasets 43 (Figure 5H). Similar OSK binding was observed in an ESC ChIP-Seq dataset albeit to a lesser extent 42 (Supplemental Figure S5C), and about one third of the cluster 2 loci have an open chromatin state in iPSCs (Supplemental Figure S5D), suggesting that some of the MORs are OSK targets in pluripotent cells.…”

“…ATAC-seq data of iPSCs were retrieved from GSE98124 41 . ChIP-seq data of ESCs and MEFs in early reprogramming at 48 hr were retrieved from GSE90895 and GSE168142, respectively 42,43 . Chip-Seq heatmaps were generated using the deepTools computeMatrix and plotHeatmap commands 62 .…”

“…ATAC-seq data of iPSCs were retrieved from GSE98124 41 . ChIP-seq data of ESCs and MEFs in early reprogramming at 48 hr were retrieved from GSE90895 and GSE168142, respectively 42,43 .…”

“…These ZFP266 binding sites have low chromatin accessibility as measured by ATAC-seq in MEFs, 72 hours after reprogramming, as well as in iPSCs 41 (Figure 4A). ZFP266 binding sites were predominantly enriched for somatic AP-1 TF motifs (Figure 4B), and little OSKM binding was observed at the same loci in 48hr reprogramming or ESC ChIP-Seq datasets 42,43 (Supplementary Figure 4B and 4C). This disputed the idea that ZFP266 functions as a suppressor at the pluripotency-related gene loci in differentiated cells.…”

Characterization of sequence determinants of enhancer function using natural genetic variation

“…Binding of TFs to their cognate motifs on nucleosomes is often restricted by steric hindrance between TFs and histone octamers. Similarly, some pioneer TFs are thought to bind partial motifs on nucleosomes (Soufi et al, 2015;Roberts et al, 2021). We considered the possibility that additional low-affinity sites, such as half motifs, make an important contribution to AP-1 binding of enhancers in fibroblasts, and that some instances of allele-specific AP-1 binding lacking core motif mutations could be explained by SNPs in degenerate recognition motifs not readily detected by traditional searches.…”