Progenitors in epithelial tissues, such as human skin epidermis, continuously make fate decisions between self-renewal and differentiation. Here we show that the Super Elongation Complex (SEC) controls progenitor fate decisions by directly suppressing a group of “rapid response” genes, which feature high enrichment of paused Pol II in the progenitor state and robust Pol II elongation in differentiation. SEC’s repressive role is dependent on the AFF1 scaffold, but not AFF4. In the progenitor state, AFF1-SEC associates with the HEXIM1-containing inactive CDK9 to suppress these rapid-response genes. A key rapid-response SEC target is ATF3, which promotes the upregulation of differentiation-activating transcription factors (GRHL3, OVOL1, PRDM1, ZNF750) to advance terminal differentiation. SEC peptidomimetic inhibitors or PKC signaling activates CDK9 and rapidly induces these transcription factors within hours in keratinocytes. Thus, our data suggest that the activity switch of SEC-associated CDK9 underlies the initial processes bifurcating progenitor fates between self-renewal and differentiation.

Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors’ regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98’s chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98’s chromatin binding and dysregulates NUP98 and RAE1’s target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.

Single-molecule localization microscopy (SMLM) enables the detailed visualization of nuclear pore complexes (NPC) in vitro with sub-20 nm resolution. However, it is challenging to translate the localized coordinates in SMLM images to NPC functions because different algorithms to cluster localizations as individual NPCs can be biased without ground truth for validation. We developed a Monte-Carlo simulation to generate synthetic SMLM images of NPC and used the simulated NPC images as the ground truth to evaluate the performance of six clustering algorithms. We identified HDBSCAN as the optimal clustering algorithm for NPC counting and sizing. Furthermore, we compared the clustering results between the experimental and synthetic data for NUP133, a subunit in the NPC, and found them to be in good agreement.

Spatial distribution of chromatin‐associated proteins provides invaluable information for understanding gene regulation. Conventional immunostaining is widely used for labeling chromatin‐associated proteins in many cell types. However, for a subset of difficult cell types, such as differentiated human keratinocytes, achieving high‐quality immunostaining for nuclear proteins remains challenging. To overcome this technical barrier, we developed the nuclei isolation staining (NIS) method. In brief, NIS involves rapid isolation of nuclei from live cells, followed by fixation and staining of the nuclei directly on coverslips for subsequent high‐magnification imaging. By removing the cytoplasmic contents and staining just the nuclei, this NIS method drastically improves antibody labeling efficiency for chromatin‐associated proteins. In this article, we describe the development and a step‐by‐step protocol of NIS, using differentiated human keratinocytes as an example. We also discuss other applications, based on the principle of this NIS method, for understanding cell‐type and cell‐state specific gene regulation. © 2019 by John Wiley & Sons, Inc.

Single-molecule localization microscopy (SMLM) enables the visualization of cellular nanostructures in vitro with sub-20 nm resolution. While substructures can generally be imaged with SMLM, the structural understanding of the images remains elusive. To better understand the link between SMLM images and the underlying structure, we developed a Monte Carlo (MC) simulation based on experimental imaging parameters and geometric information to generate synthetic SMLM images. We chose the nuclear pore complex (NPC), a nanosized channel on the nuclear membrane which gates nucleo-cytoplasmic transport of biomolecules, as a test geometry for testing our MC model. Using the MC model to simulate SMLM images, we first optimized our clustering algorithm to separate >106 molecular localizations of fluorescently labeled NPC proteins into hundreds of individual NPCs in each cell. We then illustrated using our MC model to generate cellular substructures with different angles of labeling to inform our structural understanding through the SMLM images obtained.