Abstract
The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.
Highlights
The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications
To gain insights into the role of G4s during development, we utilised a human embryonic stem cell system which enables differentiation to be studied in vitro and is an accepted model that recapitulates many aspects of cell lineage specification during human embryogenesis25. Human embryonic stem cells (hESC) were differentiated into two well-characterised, multipotent stem cells each with differing lineage potentials: cranial neural crest cells (CNCCs)[26] and neural stem cells (NSCs27, Fig. 1b, c)
We first confirmed stem cell derivation and identity using a range of established cell-lineage-specific antibody markers by immunofluorescence microscopy (IF), flow cytometry experiments and RNA-seq (Fig. 1c and Supplementary Fig. 1)
Summary
The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. HESC pluripotency is governed by a core network of master transcription factors (TFs), including OCT4, NANOG and SOX22, and their interplay with signalling pathways as well as chromatin and histone modifiers This hESC TF network sustains the pluripotent state by simultaneously promoting chromatin plasticity whilst repressing key genes involved in lineage commitment through the establishment of unique epigenetic architecture. Such changes during differentiation lead to a reorganisation of global 3D chromatin architecture to enable the activation of lineage-specific gene expression programmes and silencing of unrelated programmes[6,7,8] This is underpinned by rearrangements in promoter-enhancer interactions and super-enhancers; large clusters of regulatory elements that serve as docking sites for TFs, epigenetic modifiers and basal transcriptional machinery[6,7,8]. From cancer cell studies, that G4s modulate the functions of the (epi)genome, we present data on the potential roles of G4s in human embryonic stem cells and their differentiation into downstream lineage-specific states
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