@article {65799, title = {Dense and pleiotropic regulatory information in a developmental enhancer.}, journal = {Nature}, volume = {587}, year = {2020}, month = {2020 Nov 01}, pages = {235-39}, abstract = {

Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

}, issn = {1476-4687}, doi = {10.1038/s41586-020-2816-5}, author = {Fuqua, Timothy and Jordan, Jeff and van Breugel, Maria Elize and Halavatyi, Aliaksandr and Tischer, Christian and Polidoro, Peter and Abe, Namiko and Tsai, Albert and Mann, Richard S and Stern, David L and Crocker, Justin} } @article {49182, title = {Transvection Goes Live-Visualizing Enhancer-Promoter Communication between Chromosomes.}, journal = {Molecular Cell}, volume = {70}, year = {2018}, month = {2018 Apr 19}, pages = {195-196}, abstract = {

Lim et\ al. (2018) use live imaging in Drosophila embryos to show that enhancers can drive transcription from promoters on another chromosome when they are in close proximity. In addition, they show that multiple promoters can access the same enhancer without competition, potentially sharing a pool of factors in a transcriptional "hub."

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2018.04.004}, author = {Tsai, Albert and Singer, Robert H and Crocker, Justin} } @article {48157, title = {A fully synthetic transcriptional platform for a multicellular eukaryote.}, journal = {Cell Reports}, volume = {18}, year = {2017}, month = {2017 Jan 03}, pages = {287-296}, abstract = {

Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. Here, we explore this problem using\ a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. We found that binding sites for a transcription factor that makes DNA accessible are required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, we demonstrate that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2016.12.025}, author = {Crocker, Justin and Tsai, Albert and Stern, David L} } @article {48920, title = {Nuclear microenvironments modulate transcription from low-affinity enhancers.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Nov 02}, abstract = {

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

}, issn = {2050-084X}, doi = {10.7554/eLife.28975}, author = {Tsai, Albert and Muthusamy, Anand K and Alves, Mariana Rp and Lavis, Luke D and Singer, Robert H and Stern, David L and Crocker, Justin} } @article {47967, title = {Glutamate-induced RNA localization and translation in neurons.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, year = {2016}, month = {2016 Sep 13}, abstract = {

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

}, doi = {10.1073/pnas.1614267113}, author = {Yoon, Young J and Wu, Bin and Buxbaum, Adina R and Das, Sulagna and Tsai, Albert and English, Brian P and Grimm, Jonathan B and Lavis, Luke D and Singer, Robert H} } @article {47938, title = {Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.}, journal = {Genes and Development}, volume = {30}, year = {2016}, month = {2016 Sep 15}, pages = {2106-18}, abstract = {

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A {\textquotedblleft}step-wise{\textquotedblright} preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB{\textendash}promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II{\textendash}TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

}, doi = {10.1101/gad.285395.116}, author = {Zhang, Zhengjian and English, Brian P and Grimm, Jonathan B and Kazane, Stephanie A and Hu, Wenxin and Tsai, Albert and Inouye, Carla and You, Changjiang and Piehler, Jacob and Schultz, Peter G and Lavis, Luke D and Revyakin, Andrey and Tjian, Robert} }