Abstract
Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co‐occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome‐wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non‐dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb‐repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non‐neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells.
Highlights
Embryonic differentiation starts from a totipotent cell and culminates with the production of highly specialized cells
Capturing distinct stages of differentiation from embryonic stem cells (ESCs) to dopaminergic neurons To study the dynamic changes in Polycomb and RNA polymerase II (RNAPII) occupancy at gene promoters during differentiation, we optimized neuronal differentiation protocols to obtain large quantities of pure cell populations required for mapping chromatin-associated histone marks and RNAPII at five states of neuronal differentiation that leads to the production of functional dopaminergic neurons (ESC, days 1, 3, 16, and 30; Fig 1A)
Most genes repressed by Polycomb in ESCs are kept in a poised state through their association with an unusual form of RNAPII characterized by the S5p modification (Stock et al, 2007; Brookes et al, 2012; Tee et al, 2014; Ma et al, 2016)
Summary
Embryonic differentiation starts from a totipotent cell and culminates with the production of highly specialized cells. In ESCs, many genes important for early development are repressed in a state that is poised for subsequent activation (Azuara et al, 2006; Bernstein et al, 2006; Stock et al, 2007; Brookes et al, 2012) These genes are mostly GC-rich (Deaton & Bird, 2011), and their silencing in pluripotent cells is mediated by Polycomb repressive complexes (PRCs). Polycomb repressive complex proteins have major roles in modulating gene expression during differentiation and in disease (Prezioso & Orlando, 2011; Richly et al, 2011) They assemble in two major complexes, PRC1 and PRC2, which catalyze H2AK119 monoubiquitination and H3K27 methylation, respectively (Simon & Kingston, 2013). Both PRC-mediated histone marks are important for chromatin repression, and synergize in a tight feedback loop to recruit each other’s modifying enzymes (Blackledge et al, 2015)
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