Transcription Of Endogenous Genes Research Articles

A simple MiMIC based approach for tagging endogenous genes to visualise live transcription in Drosophila.

Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem loops into endogenous genes requires laborious CRISPR genome editing. Here we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging and live imaging transcription of endogenous genes and the long non-coding RNA, roX1, in the embryo. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues.

A digital CRISPR-dCas9-based gene remodeling biocomputer programmed by dietary compounds in mammals

CRISPR-dCas9 (dead Cas9 protein) technology, combined with chemical molecules and light-triggered genetic switches, offers customizable control over gene perturbation. However, these simple ON/OFF switches cannot precisely determine the sophisticated perturbation process. Here, we developed a resveratrol and protocatechuic acid-programmed CRISPR-mediated gene remodeling biocomputer (REPACRISPR) for conditional endogenous transcriptional regulation of genes invitro and invivo. Two REPACRISPR variants, REPACRISPRi and REPACRISPRa, were designed for the logic control of gene inhibition and activation, respectively. We successfully demonstrated the digital computations of single or multiplexed endogenous gene transcription by using REPACRISPRa. We also established mathematical models to predict the dose-responsive transcriptional levels of a target endogenous gene controlled by REPACRISPRa. Moreover, high levels of endogenous gene activation in mice mediated by the AND logic gate demonstrated computational control of CRISPR-dCas9-based epigenome remodeling in mice. This CRISPR-based biocomputer expands the synthetic biology toolbox and can potentially advance gene-based precision medicine. A record of this paper's transparent peer review process is included in the supplemental information.

Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system.

The recently developed CRISPR activator (CRISPRa) system uses a CRISPR-Cas effector-based transcriptional activator to effectively control the expression of target genes without causing DNA damage. However, existing CRISPRa systems based on Cas9/Cas12a necessitate improvement in terms of efficacy and accuracy due to limitations associated with the CRISPR-Cas module itself. To overcome these limitations and effectively and accurately regulate gene expression, we developed an efficient CRISPRa system based on the small CRISPR-Cas effector Candidatus Woesearchaeota Cas12f (CWCas12f). By engineering the CRISPR-Cas module, linking activation domains, and using various combinations of linkers and nuclear localization signal sequences, the optimized eCWCas12f-VPR system enabled effective and target-specific regulation of gene expression compared with that using the existing CRISPRa system. The eCWCas12f-VPR system developed in this study has substantial potential for controlling the transcription of endogenous genes in living organisms and serves as a foundation for future gene therapy and biological research.

MTOR Regulates Mineralocorticoid Receptor Transcriptional Activity by ULK1-Dependent and -Independent Mechanisms.

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.

CRISPR-Cas13-mediated RNA editing in the silkworm Bombyx mori.

The CRISPR-Cas13 system, an RNA-guided editing tool, has emerged as a highly efficient and stable RNA editing technique. Although the CRISPR-Cas13 system has been developed in several insect species, its application in lepidopterans has not yet been reported. In the present study, we evaluated the RNA cleavage activity of the CRISPR-Cas13 system in the silkworm ( Bombyx mori), a model lepidopteran insect, both ex vivo and in vivo. We established two stable silkworm BmE cell lines expressing PspCas13b and CasRx, respectively. Further analysis demonstrated that both PspCas13b and CasRx effectively down-regulated the transcription of exogenously-introduced target and endogenous genes in these cell lines. In addition, we generated two transgenic silkworm strains, one expressing CasRx and the other expressing RNA-guided CRISPR RNA targeting Sex combs reduced ( Scr). Further crossing experiments showed that CasRx induced a down-regulation of Scr transcription in silkworms, which impaired systemic growth of larvae. Overall, this study demonstrated that the CRISPR-Cas13 RNA editing system works efficiently in the silkworm, providing a potential alternative approach for RNA manipulation in lepidopteran insects.

Design of hypoxia responsive CRISPR-Cas9 for target gene regulation

The CRISPR–Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR–Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.

Light-triggered site-directed RNA editing by endogenous ADAR1 with photolabile guide RNA

RNA A-to-I editing is a post-transcriptional modification pervasively occurring in cells. Artificial intervention of A-to-I editing at specific sites of RNA could also be achieved with guide RNA and exogenous ADAR enzymes. In contrast to previous fused SNAP-ADAR enzymes for light-driven RNA A-to-I editing, we developed photo-caged antisense guide RNA oligonucleotides with simple 3'-terminal cholesterol modification, and successfully achieved light-triggered site-specific RNA A-to-I editing for the first time utilizing endogenous ADAR enzymes. Our caged A-to-I editing system effectively implemented light-dependent point mutation of mRNA transcripts of both exogenous and endogenous genes in living cells and 3D tumorspheres, as well as spatial regulation of EGFP expression, which provides a new approach for precise manipulation of RNA editing.

African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

This study evaluates the role of the late viral protein, pA104R, in African swine fever virus immunosuppression. ASFV-encoded pA104R is a putative histone-like protein that is highly conserved throughout different virulent and non-virulent isolates. Previous studies have demonstrated that pA104R plays a vital role in the ASFV replication cycle and is a potential target for antiviral therapy. Here, we demonstrated that pA104R is a potent antagonist of type I interferon signaling. IFN-stimulated response element activity and subsequent transcription of co-transfected and endogenous interferon-stimulated genes were attenuated by pA104R treatment in HEK-293 T cells. Immunoprecipitation assay and reciprocal pull-down showed that pA104R does not interact directly with STAT1, STAT2, or IRF9. However, pA104R could inhibit IFN signaling by attenuating STAT1 phosphorylation, and we identified the critical amino acid residues (R/H69,72 and K/R92,94,97) involved through the targeted mutation functional assays. Although pA104R is a histone-like protein localized to the nucleus, it did not inhibit IFN signaling through its DNA-binding capacity. In addition, activation of the ISRE promoter by IRF9-Stat2(TA), a STAT1-independent pathway, was inhibited by pA104R. Further results revealed that both the transcriptional activation and recruitment of transcriptional stimulators by interferon-stimulated gene factor 3 were not impaired. Although we failed to determine a mechanism for pA104R-mediated IFN signaling inhibition other than attenuating the phosphorylation of STAT1, these results might imply a possible involvement of epigenetic modification by ASFV pA104R. Taken together, these findings support that pA104R is an antagonist of type I interferon signaling, which may interfere with multiple signaling pathways.

Light-activated macromolecular phase separation modulates transcription by reconfiguring chromatin interactions.

Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.

A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.

Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.

Abstract IA019: Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription

Abstract Mammalian transcription factors (TFs) ubiquitously contain intrinsically disordered low-complexity domains (LCDs) that perform transactivation functions. Recent studies have shown that dynamic, multivalent, and selective interactions between TF LCDs play an essential role in transactivation, but how such interactions mediate gene transcription remains unclear. It has been proposed that liquid-liquid phase separation (LLPS) of TFs driven by extensive LCD-LCD interactions is the mechanism underlying transactivation. Here, we investigate how tuning the level and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. By quantitative single-cell imaging, we reveal that efficient gene activation by the oncogenic TF EWS/FLI1 requires narrow optimum of LCD-LCD interactions at its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in Ewing sarcoma cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a well-documented LLPS compartment, the nucleolus, represses EWS/FLI1 -driven transcription and inhibits malignant transformation of Ewing sarcoma. In addition, using live-cell single-molecule imaging, we show that a TF diffuses significantly more slowly in the nucleolus than in the nucleoplasm, demonstrating an important impact of LLPS on TF dynamic behaviors and single-molecule diffusion measurement as an effective approach to diagnose LLPS in vivo. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs. Citation Format: Shasha Chong. Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA019.

Recent Progress and Future Prospect of CRISPR/Cas-Derived Transcription Activation (CRISPRa) System in Plants.

Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed.

Altering enhancer-promoter linear distance impacts promoter competition in cis and in trans.

In Drosophila, pairing of maternal and paternal homologs can permit trans-interactions between enhancers on one homolog and promoters on another, an example of a phenomenon called transvection. When chromosomes are paired, promoters in cis and in trans to an enhancer can compete for the enhancer's activity, but the parameters that govern this competition are as yet poorly understood. To assess how the linear spacing between an enhancer and promoter can influence promoter competition in Drosophila, we employed transgenic constructs wherein the eye-specific enhancer GMR is placed at varying distances from a heterologous hsp70 promoter driving a fluorescent reporter. While GMR activates the reporter to a high degree when the enhancer and promoter are spaced by a few hundred base pairs, activation is strongly attenuated when the enhancer is moved 3kb away. By examining transcription of endogenous genes near the point of transgene insertion, we show that linear spacing of 3 kb between GMR and the hsp70 promoter results in elevated transcription of neighboring promoters, suggesting a loss of specificity between the enhancer and its intended transgenic target promoter. Furthermore, increasing spacing between GMR and hsp70 by just 100 bp can enhance transvection, resulting in increased activation of a promoter on a paired homolog at the expense of a promoter in cis to the enhancer. Finally, cis-/trans-promoter competition assays in which one promoter carries mutations to key core promoter elements show that GMR will skew its activity toward a wild-type promoter, suggesting that an enhancer is in a balanced competition between its potential target promoters in cis and in trans.

Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription

Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions invivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.

Gene activation in Caenorhabditis elegans using the Campylobacter jejuni CRISPR-Cas9 feeding system.

Clustered regularly interspaced palindromic repeats-based activation system, a powerful genetic manipulation technology, can modulate endogenous gene transcription in various organisms through fusing nuclease-deficient Cas9 to transcriptional regulatory domains. At present, this clustered regularly interspaced palindromic repeats-based activation system has been applied to activate gene expression by microinjection manner in Caenorhabditis elegans. However, this complicated and time-consuming injection manner is not suitable for efficient and high-throughput gene regulation with clustered regularly interspaced palindromic repeats-Cas9 system. Here, we engineered a Campylobacter jejun clustered regularly interspaced palindromic repeats-Cas9-based gene activation system through bacteria feeding technique to delivering gene-specific sgRNA in C. elegans. It enables to activate various endogenous genes efficiently, as well as induce the corresponding phenotypes with a more efficient and labor-saving manner. Collectively, our results demonstrated that our novel dCjCas9-based activation feeding system holds great promise and potential in C. elegans.

CRISPR Interference as a Tool to Repress Gene Expression in Haloferax volcanii.

To date, a plethora of tools for molecular biology have been developed on the basis of the CRISPR-Cas system. Almost all use the class 2 systems since here the setup is the simplest with only one protein and one guide RNA, allowing for easy transfer to and expression in other organisms. However, the CRISPR-Cas components harnessed for applications are derived from mesophilic bacteria and are not optimal for use in extremophilic archaea.Here, we describe the application of an endogenous CRISPR-Cas system as a tool for silencing gene expression in a halophilic archaeon. Haloferax volcanii has a CRISPR-Cas system of subtype I-B, which can be easily used to repress the transcription of endogenous genes, allowing to study the effects of their depletion. This article gives a step-by-step introduction on how to use the implemented system for any gene of interest in Haloferax volcanii. The concept of CRISPRi described here for Haloferax can be transferred to any other archaeon, that is genetically tractable and has an endogenous CRISPR-Cas I systems.

Delivery of the VIVIT Peptide to Human Glioma Cells to Interfere with Calcineurin-NFAT Signaling.

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.

Estrogen Receptor-Alpha and p53 Status as Regulators of AMPK and mTOR in Luminal Breast Cancer.

Simple SummaryEndocrine therapy targeted against estrogen and the estrogen receptor is the main treatment modality for luminal breast cancer. Although a majority of patients respond to this treatment, one-third of all patients are resistant to this therapy. Hyperactive estrogen receptor along with alternative drivers such as mutations in the major tumor suppressor named p53 are known to contribute to resistance to therapy. The current study shines light on some of the mechanisms underlying the functioning of these players. A better understanding of these mechanisms will have important therapeutic implications by facilitating development of strategies to overcome therapeutic resistance by identifying novel targets and by combining drugs to effectively combat luminal breast cancer.Luminal breast cancer (LBC) driven by dysregulated estrogen receptor-alpha (ERα) signaling accounts for 70% of the breast cancer cases diagnosed. Although endocrine therapy (ET) is effective against LBC, about one-third of these patients fail to respond to therapy owing to acquired or inherent resistance mechanisms. Aberrant signaling via ERα, oncogenes, growth factor receptors, and mutations in tumor suppressors such as p53 impinge on downstream regulators such as AMPK and mTOR. While both AMPK and mTOR have been reported to play important roles in determining sensitivity of LBC to ET, how the ERα-p53 crosstalk impinges on regulation of AMPK and mTOR, thereby influencing therapeutic efficacy remains unknown. Here, we have addressed this important issue using isogenic breast cancer cell lines, siRNA-mediated RNA knockdown, and different modes of drug treatments. Interaction of p53 with ERα and AMPK was determined by in situ proximity ligation assay (PLA), and endogenous gene transcripts were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Further, the effect of concurrent and sequential administration of Fulvestrant–Everolimus combination on colony formation was determined. The studies showed that in cells expressing wild type p53, as well as in cells devoid of p53, ERα represses AMPK, whereas in cells harboring mutant p53, repression of AMPK is sustained even in the absence of ERα. AMPK is a major negative regulator of mTOR, and to our knowledge, this is the first study on the contribution of AMPK-dependent regulation of mTOR by ERα. Furthermore, the studies revealed that independent of the p53 mutation status, combination of Fulvestrant and Everolimus may be a viable first line therapeutic strategy for potentially delaying resistance of ERα+/HER2− LBC to ET.

Intron removal by the spliceosome: A solo job or a team effort?

Wan etal. (2021) establish a powerful new platform to measure the dynamics of transcription and splicing of endogenous genes in single cells in real time. Combining real-time measurements with multiple deep-sequencing tools reveals an unexpectedly high amount of spliceosome activity, prompting a reconsideration of current models of how introns are removed from pre-mRNA.

Approaches to Dissect the Role of Endosome Positioning in Spatially Encoded GPCR Signaling

G protein-coupled receptors (GPCRs), which comprise the largest class of drug targets, are critical signaling proteins that translate extracellular stimuli to mediate human physiology. As such, understanding GPCR signaling cascades will provide insight into the molecular processes underlying complex physiologies, how these are perturbed in disease, and inform our ability to design more efficient therapeutics. In recent years, my lab and others have made the paradigm-shifting discovery that GPCRs can be activated after drug-induced internalization from the plasma membrane into endosomal compartments to generate spatially biased responses. We have found that transcriptional responses to GPCR activation are initiated strictly from intracellular receptors. However, it remains unknown how the endosome promotes these selective signaling outcomes. One possibility is that the biophysical proximity of endosomal GPCRs to the nucleus allow for cAMP signaling cascades that result in gene transcription. To address this, I have devised a strategy to rapidly redistribute endosomes towards the cell periphery and coupled it with microscopy-based transcriptional readouts. Specifically, I have optimized a chemically induced heterodimerization system that fuses endosomal embedding proteins to plus- or minus-end directed molecular motors. To determine the impact of endosomal redistribution on transcription, an endosomal selective GPCR response, I monitor accumulation of a fluorescent cAMP transcriptional reporter and use in situ RNA hybridization to examine transcription of endogenous cAMP target genes. I will discuss recent progress made with these approaches to dissect how spatial organization of endosomes influences downstream GPCR signaling. Ultimately, this work will help dissect what makes the endosome a unique signaling compartment and illuminate the molecular mechanisms underlying spatial selectivity of GPCR signaling.