Recent evidence that TADs and chromatin loops are dynamic structures.

Mammalian genomes are folded into spatial domains, which regulate gene expression by modulating enhancer-promoter contacts. Here, we review recent studies on the structure and function of Topologically Associating Domains (TADs) and chromatin loops. We discuss how loop extrusion models can explain TAD formation and evidence that TADs are formed by the ring-shaped protein complex, cohesin, and that TAD boundaries are established by the DNA-binding protein, CTCF. We discuss our recent genomic, biochemical and single-molecule imaging studies on CTCF and cohesin, which suggest that TADs and chromatin loops are dynamic structures. We highlight complementary polymer simulation studies and Hi-C studies employing acute depletion of CTCF and cohesin, which also support such a dynamic model. We discuss the limitations of each approach and conclude that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle

Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers.

In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation

A dynamic mode of mitotic bookmarking by transcription factors.

During mitosis, transcription is shut off, chromatin condenses, and most transcription factors (TFs) are reported to be excluded from chromosomes. How do daughter cells re-establish the original transcription program? Recent discoveries that a select set of TFs remain bound on mitotic chromosomes suggest a potential mechanism for maintaining transcriptional programs through the cell cycle termed mitotic bookmarking. Here we report instead that many TFs remain associated with chromosomes in mouse embryonic stem cells, and that the exclusion previously described is largely a fixation artifact. In particular, most TFs we tested are significantly enriched on mitotic chromosomes. Studies with Sox2 reveal that this mitotic interaction is more dynamic than in interphase and is facilitated by both DNA binding and nuclear import. Furthermore, this dynamic mode results from lack of transcriptional activation rather than decreased accessibility of underlying DNA sequences in mitosis. The nature of the cross-linking artifact prompts careful re-examination of the role of TFs in mitotic bookmarking

Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization

Robust model-based analysis of single-particle tracking experiments with Spot-On.

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce 'Spot-On', an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants

A stable mode of bookmarking by TBP recruits RNA polymerase II to mitotic chromosomes.

Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos.

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.Editorial noteThis article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)