Nuclear myosin 1 (NM1) has been implicated in key nuclear functions. Together with actin, it has been shown to initiate and regulate transcription, it is part of the chromatin remodeling complex B-WICH, and is responsible for rearrangements of chromosomal territories in response to external stimuli. Here we show that deletion of NM1 in mouse embryonic fibroblasts leads to chromatin and transcription dysregulation affecting the expression of DNA damage and cell cycle genes. NM1 KO cells exhibit increased DNA damage and changes in cell cycle progression, proliferation, and apoptosis, compatible with a phenotype resulting from impaired p53 signaling. We show that upon DNA damage, NM1 forms a complex with p53 and activates the expression of checkpoint regulator p21 (Cdkn1A) by PCAF and Set1 recruitment to its promoter for histone H3 acetylation and methylation. We propose a role for NM1 in the transcriptional response to DNA damage response and maintenance of genome stability.

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators ( NUP93 , PRIM1 , RUVBL1 , PKMYT1 , TP53 , SF3A2 , and HRAS ), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and weaker phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with weaker phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.Author summaryCells sense and respond to changes in their surrounding environment through G protein-coupled receptors (GPCRs) and their associated cascades. The proper function of these pathways is essential to human physiology, and GPCRs have become a prime target for drug development for a range of human diseases. Therefore, it is of utmost importance to be able to map how these pathways operate to enable cells to fine-tune their responsiveness. Here, we describe a screening approach that we have devised to systematically identify regulators of GPCR function. We have developed a sortable reporter system and coupled that with silencing of genes across the entire human genome in order to uncover a range of novel mediators of GPCR activity. We characterize a few of these new regulators and show that they function at different steps of the cascade. Therefore, this study serves as proof of principle for the new screening platform. We envision that the approach can be used to dissect additional dimensions of GPCR function, including regulators of drug-specific responses, functional characterization of receptor features, and identification of novel drugs, and thus advance a genome-scale understanding of these critical pathways.

No abstract

The beta‐adrenergic (β2‐AR) receptor is a prototypical GPCR that controls important functions in the nervous, pulmonary, and cardiovascular systems. β2‐AR signaling involves multiple factors in both G‐protein dependent and independent signaling, resulting in diverse signaling outcomes. To understand the complexity of this pathway, we have performed the first unbiased, high‐throughput functional genomics screen of β2‐AR signaling using cAMP reporter and CRISPR interference (CRISPRi) silencing. We generated a reporter cell line using a construct encoding cAMP‐response element (CREB) fused to a ProteoTuner destabilizing domain and green fluorescent protein and another construct encoding catalytically‐dead Cas9 (dCas9) fused to the transcriptional repressor KRAB. Reporter cells were transduced with genome‐wide lentiviral sgRNA library to silence individual genes. After induction of β2‐AR signaling, we sorted the edited cells based on reporter expression and subjected them to deep sequencing and statistical analysis of sgRNA enrichment. We obtained 32 positive and 14 negative β2‐AR/cAMP signaling modulators enriched in factors involved in transcription initiation, RNA splicing, and RNA metabolic processes. We validated TP53 and NUP93 as novel positive modulators of β2‐AR signaling using dual luciferase cAMP assay and RT‐qPCR of cAMP‐dependent target gene, consistent with our cAMP reporter result. We found that the TP53‐dependent modulation of this pathway can partially be explained by lower transcription and surface expression of the β2‐AR receptors. These findings demonstrate a successful application of our reporter in a high‐throughput approach to identify previously uncharacterized modulators of the β2‐AR/cAMP pathway. This study reveals broader potential applications of our cAMP reporter as a platform to understand other aspects of GPCR signal transduction such as biased signaling, receptor‐specific molecular mechanisms, and drug discovery. Support or Funding Information This work is supported by the National Institute of Mental Health R00 award (MH109633) awarded to Nikoleta Tsvetanova.

No abstract

G protein‐coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome‐wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow‐up experiments, we validated the functional effects of eight newly discovered positive and negative mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, HRAS, RBM12), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high‐throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.

G‐protein coupled receptors (GPCRs) are pivotal to neuronal functions such as neurotransmission and memory formation, and aberrant GPCR signaling has been implicated in complex neuropsychiatric disorders. Our ability to develop efficient therapeutic strategies therefore hinges on better understanding of the molecular factors that govern GPCR activity. Upon activation, GPCRs modulate a myriad of downstream second messengers and molecules that generate cellular responses, including the transcription of cAMP‐dependent target genes. Through a genome‐wide CRISPR‐based screen with a fluorescent cAMP transcriptional reporter, we identified RNA‐binding motif 12 (Rbm12) as a novel potent repressor of the GPCR/cAMP pathway. While Rbm12 is a genetic risk factor for familial psychosis, its precise cellular functions are unknown. We hypothesize that the loss of Rbm12’s regulation of the GPCR/cAMP pathway contributes to the development of psychosis. To investigate this intriguing possibility, we performed CRISPR‐based gene editing to show that Rbm12 deficiency leads to increased cAMP accumulation and hyperactivation of multiple cAMP‐dependent target genes downstream of several key neurotransmitter GPCRs. The signaling hyperactivation caused by Rbm12 loss is similarly conserved in human induced pluripotent stem cell‐derived neurons. Furthermore, we demonstrate that disease‐linked truncating mutations in Rbm12 (c.2377G>T and c.2532delT) fail to rescue the signaling hyperactivation due to possible loss‐of‐function and protein instability, respectively. Together, these experiments provide critical insights into this uncharacterized gene. By uncovering the novel regulatory role of a neuropsychiatric disease risk gene in GPCR signaling, we expand our understanding of the molecular basis of complex neurobiological disorders and enable the identification of novel druggable targets.