Abstract
The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.
Highlights
Transcription factors (TFs) regulate the expression of genes through interactions with specific DNA sequences located in gene promoters and distal regulatory elements
Loci that lost accessibility were highly enriched for OCT4 and SOX2 ChIP-seq binding while loci that gained accessibility were much less so (Fig.S2A-B). To compare the loci impacted by OCT4 vs SOX2 depletion, we focused on all regions that were bound by OCT4 and/or SOX2 as identified from available and newly generated ChIP-seq datasets and that lost accessibility upon dox treatment
The OCT4 ChIP-seq signal was correlated to loss of accessibility upon OCT4 depletion (Fig. S8A) as shown previously, and to chromatin accessibility in untreated cells (Fig. S8B), indicating that strong OCT4 binding sites are both highly accessible and sensitive to OCT4 levels. These results reveal different classes of OCT4-bound loci that show different cell cycle accessibility dynamics upon OCT4 loss at the M-G1 transition, and that highly bound sites are accessible and sensitive to OCT4 loss for the maintenance of their accessibility and H3K27 acetylation. 373 OCT4 is required throughout the cell cycle to maintain enhancer accessibility We asked whether OCT4 plays a role in maintaining enhancer accessibility in other cell cycle phases
Summary
Transcription factors (TFs) regulate the expression of genes through interactions with specific DNA sequences located in gene promoters and distal regulatory elements. Much less is known about the role of pioneer activity and of its dynamics over the cell cycle in regulating stem cell self-renewal. Self-renewal requires the ability to progress through the cell cycle without losing cell type[52] specific gene expression. This is not a trivial task since chromatin accessibility of gene regulatory elements is markedly decreased during S phase and mitosis (Festuccia et al, 2019; Hsiung et al, 2015; Oomen et al, 2019; Stewart-Morgan et al, 2019). OCT4 expression levels in G1 phase affect the propensity of ES cells to differentiate towards neuroectoderm and mesendoderm (Strebinger et al, 2019), and depletion of OCT4 at the M-G1 transition impairs pluripotency maintenance of ES cells and leads to a lower reprogramming efficiency upon overexpression in mouse embryonic fibroblasts (Liu et al, 2017). Using forms of OCT4 engineered for mitotic or auxin-inducible degradation, we demonstrate the role of OCT4 in re[76] establishment and continuous maintenance of chromatin accessibility throughout the cell cycle. 79
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