SignificanceThis study compares the native structures of cytosolic and nuclear proteasomes, visualized directly within cells. The assembly states and functional states of proteasomes in each compartment were similar, indicating comparable levels of proteolytic activity per proteasome. Nuclear proteasomes were tethered to two different sites at the nuclear pore complex (NPC): the inner nuclear membrane and the NPC basket. Structural analysis revealed mechanistic details of the two tethering interactions. These results present direct evidence that proteasomes bind at NPCs, establishing a cellular hub for protein degradation at the gateway between the nucleus and cytoplasm. This work demonstrates how cryo-electron tomography can reveal biological mechanisms by directly observing the interactions between molecular complexes within the native cellular environment.

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large‐scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre‐ to post‐implantation epiblast in utero. We identified 496 naïve state‐associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition.

Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. Yet, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that form a previously identified enhancer cluster and activate the Fgf5 locus during exit from naïve murine pluripotency. Four elements are located downstream of the Fgf5 gene and form a super-enhancer. Each of these elements contributes to Fgf5 induction at a distinct time point of differentiation. The fifth element is located in the first intron of the Fgf5 gene and contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. This amplifier element strongly accumulates paused RNA Polymerase II but does not give rise to a mature Fgf5 mRNA. By transplanting the amplifier to a different genomic position, we demonstrate that it enriches for high levels of paused RNA Polymerase II autonomously. Based on our data, we propose a model for a mechanism by which RNA Polymerase II accumulation at a novel type of enhancer element, the amplifier, contributes to enhancer collaboration..

AbstractIn the mammalian embryo, epiblast cells must exit their naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene-networks involved in the exit from naïve pluripotency remains fragmented. Here we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems-biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 knockout ESC lines predominantly manifest delays on the trajectory from naive to formative epiblast. We find that gene networks operative in ESCs are active during transition from pre- to post-implantation epiblast in utero. We identified 374 naïve-associated genes tightly connected to epiblast state and largely conserved in human ESCs and primate embryos. Integrated analysis of mutant transcriptomes revealed funneling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signaling pathways direct this pivotal mammalian cell state transition.

14Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. Yet, 15how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the 16 functions and interdependencies of five enhancer elements that form a previously identified enhancer 17 cluster and activate the Fgf5 locus during exit from naïve murine pluripotency. Four elements are located 18 downstream of the Fgf5 gene and form a super-enhancer. Each of these elements contributes to Fgf5 19 induction at a distinct time point of differentiation. The fifth element is located in the first intron of the 20 Fgf5 gene and contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression 21 levels. This amplifier element strongly accumulates paused RNA Polymerase II but does not give rise to a 22 mature Fgf5 mRNA. By transplanting the amplifier to a different genomic position, we demonstrate that it 23 enriches for high levels of paused RNA Polymerase II autonomously. Based on our data, we propose a 24 model for a mechanism by which RNA Polymerase II accumulation at a novel type of enhancer element, 25 the amplifier, contributes to enhancer collaboration. 26

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Tight control of the transcription process is essential for the correct spatial and temporal gene expression pattern during development and in homeostasis. Enhancers are at the core of correct transcriptional activation. The original definition of an enhancer is straightforward: a DNA sequence that activates transcription independent of orientation and direction. Dissection of numerous enhancer loci has shown that many enhancer‐like elements might not conform to the original definition, suggesting that enhancers and enhancer‐like elements might use multiple different mechanisms to contribute to transcriptional activation. Here, we review methodologies to identify enhancers and enhancer‐like elements and discuss pitfalls and consequences for our understanding of transcriptional regulation.