Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus (“localization-resets”) activates YAP target genes. These “resets” are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.
The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.
The promiscuity of G-protein-coupled receptors (GPCRs) has broad implications in disease, pharmacology and biosensing. Promiscuity is a particularly crucial consideration for protein engineering, where the ability to modulate and model promiscuity is essential for developing desirable proteins. Here, we present methodologies for (i) modifying GPCR promiscuity using directed evolution and (ii) predicting receptor response and identifying important peptide features using quantitative structure-activity relationship models and grouping-exhaustive feature selection. We apply these methodologies to the yeast pheromone receptor Ste2 and its native ligand α-factor. Using directed evolution, we created Ste2 mutants with altered specificity toward a library of α-factor variants. We then used the Vectors of Hydrophobic, Steric, and Electronic properties and partial least squares regression to characterize receptor-ligand interactions, identify important ligand positions and properties, and predict receptor response to novel ligands. Together, directed evolution and computational analysis enable the control and evaluation of GPCR promiscuity. These approaches should be broadly useful for the study and engineering of GPCRs and other protein-small molecule interactions.
Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, we report a robust experimental platform for real-time visualization of native YAP dynamics and target gene expression. Using this platform, we show that activation of YAP target genes is preceded by concerted localization resets, which are dramatic, concerted departure/reentry cycles of nuclear YAP. These resets could be induced by calcium signaling, acto-myosin contractility, and mitotic-exit, and were strictly correlated with YAP-dependent transcription measured using nascent-transcription reporter knockins of YAP target genes. Oncogenically transformed cells with chronically elevated YAP-driven transcription lacked resets but rapidly exchanged YAP between the nucleus and cytoplasm, suggesting an escape from compartmentalization-based control. The single-cell YAP localization and transcription traces suggest a new mode of transcriptional regulation involving the concerted re-partitioning of YAP prior to gene activation. Results and DiscussionThe YAP (YES-associated protein) / TAZ (transcriptional coactivator with PDZ-binding motif) duo(1), a central node of the Hippo pathway (2)(3), controls organ size, and is critical for mechanotransduction (4)(5)(6). The classic view of YAP signal transduction equates nuclear enrichment with activation of pro-growth transcriptional programs through association with the TEAD family of transcription factors (7) (8) (9). However, other critical signal transducers such as ERK, NfkB and P53 use a rich signal transmission code in which the amplitude, frequency, and duration of their nucleocytoplasmic shuttling all influence gene transcription(10)(11)(12). Given recent reports that both YAP and TEAD can alter subcellular localization(13)(14)(15)(16), we speculated that their subcellular spatiotemporal dynamics may encode upstream signaling information. More generally, we should not expect biological signal transmission circuits to be simple, e.g. a linear mapping between an external input onto downstream gene expression level. There are now many examples of gene circuits with hysteresis, memory, and latching (17)(18)(19)(20)(21). Since these circuits involve spatially-controlled components such as transcription factors, it is natural to wonder about the interplay of cellular spatial dynamics and signal processing.To investigate these ideas, we sought to quantify the localization dynamics of native YAP/TEAD in single cells and relate such dynamics to downstream transcription. We used CRISPR(22) to fluorescently tag native YAP and TEAD in breast epithelial cell lines commonly used for studying hippo signaling(23) (Methods, Figure 1a). Since transcription is pulsatile(24)(25), relating localization dynamics to target gene activity requires real-time measurement of gene transcription. We therefore used CRISPR to tag the native mRNAs of two classic YAP/TEAD targets, ANKRD1 (26) and AREG (27), with 24x-MS2 transcriptional reporters.A recen...
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