A role for mitotic bookmarking of SOX2 in pluripotency and differentiation

Abstract

Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.

Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells

“…UTF1, a chromatin-associated factor with histone-like characteristics (Kooistra et al, 2009; van den Boom et al, 2007), served as a positive control showing almost identical levels in asynchronous and mitotic chromatin extracts (Figures 1D–E). In agreement with recent studies (Festuccia et al, 2016; Teves et al, 2016; Deluz et al, 2016), this assay showed that SOX2, ESRRB and OCT4 as well as KLF4 were also highly retained on mitotic chromatin (ranging between 70–100% of respective levels in asynchronous cells). By contrast, factors such as NANOG and REX1 showed markedly reduced levels in the chromatin fraction during mitosis (Figures 1D–E).…”

“…This finding was surprising given that previously reported bookmarked targets by various TFs, including the recently described ESRRB and SOX2 in ESCs (Festuccia et al, 2016; Deluz et al, 2016), are often characterized by weaker binding during mitosis, suggesting either technical or biological differences between studies. To test the possibility that the strength of the detected signal in our mitotic samples was due to contamination from residual G2 cells (not more than 10% in our mitotic samples), we mixed interphase ESCs and neuronal progenitor cells (NPCs), which do not express KLF4, in a ratio of 10:90 and performed KLF4 ChIP-seq and ChIP-qPCR.…”

“…Here, using multiple independent experimental approaches, we report that a large number of stem cell regulators, including the recently described ESRRB and SOX2 (Festuccia et al, 2016; Deluz et al; 2016), were largely retained on mitotic chromatin of ESCs. Interestingly, in contrast with these studies, our data revealed a strong, widespread-yet-specific binding of KOS during mitosis.…”

“…Among them, OCT4, SOX2, ESRRB and KLF4 have been reported to also function as pioneer factors (Iwafuchi-Doi and Zaret, 2014; Soufi et al, 2012; Soufi et al, 2015), sharing critical properties with known bookmarking TFs. However, evidence for a potential bookmarking role for some of these TFs has only recently begun to emerge (Festuccia et al, 2016; Teves et al, 2016; Deluz et al, 2016). Similarly, although the functional genomic elements that control stem cell identity and the histone marks that decorate them are well characterized (Hawkins et al, 2010; Mikkelsen et al, 2007; Whyte et al, 2013), their status during mitosis remains unknown.…”

Pioneer transcription factors shape the epigenetic landscape

“…Both specific and non-specific binding sites on mitotic chromosomes were also observed for FoxA1 (77) where specific FoxA1 binding occurs at 15% of its interphase targets. Recently, Sox2 and Oct4 were also shown to remain bound during mitosis (78,79). In this last study, the authors also show using live imaging techniques that crosslinking with formaldehyde leads to eviction of most TFs from mitotic chromosomes.…”