CIAO1 and MMS19 deficiency: A lethal neurodegenerative phenotype caused by cytosolic Fe-S cluster protein assembly disorders.

PURPOSE: The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system. METHODS: Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences. RESULTS: Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models. CONCLUSION: A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

phox2ba: The Potential Genetic Link behind the Overlap in the Symptomatology between CHARGE and Central Congenital Hypoventilation Syndromes.

CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7). CHD7 is involved in regulating neural crest development, which gives rise to tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of PHOX2B (paired-like homeobox 2b) underlies CCHS. Employing a chd7-null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of phox2b. Heart rates were lower in chd7 mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that chd7 mutants took longer to become anesthetized, with higher respiratory rates during recovery. chd7 mutant larvae demonstrated unique phox2ba expression patterns. The knockdown of phox2ba reduced larval heart rates similar to chd7 mutants. chd7 mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS.

Loss of calpain3b in Zebrafish, a Model of Limb-Girdle Muscular Dystrophy, Increases Susceptibility to Muscle Defects Due to Elevated Muscle Activity.

Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3. In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20-30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1.

KIT D816V is dimerization-independent and activates downstream pathways frequently perturbed in mastocytosis.

KIT, a type III tyrosine kinase receptor, plays a crucial role in haematopoietic development. The KIT receptor forms a dimer after ligand binding; this activates tyrosine kinase activity leading to downstream signal transduction. The D816V KIT mutation is extensively implicated in haematological malignancies, including mastocytosis and leukaemia. KIT D816V is constitutively active, but the molecular nuances that lead to constitutive tyrosine kinase activity are unclear. For the first time, we present experimental evidence that the KIT D816V mutant does not dimerize like KIT wild type. We further show evidence of decreased stabilization of the tyrosine kinase domain in the KIT D816V mutant, a phenomenon that might contribute to its constitutive activity. Since the mechanism of KIT D816V activation varies from that of the wild type, we explored downstream signal transduction events and found that even though KIT D816V targets similar signalling moieties, the signalling is amplified in the mutant compared to stem cell factor-activated wild type receptor. Uniquely, KIT D816V induces infection-related pathways and the spliceosome pathway, providing alternate options for selective as well as combinatorial therapeutic targeting.

Human JAK1 gain of function causes dysregulated myelopoeisis and severe allergic inflammation.

Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.

Zebrafish models of inflammation in hematopoietic development and disease.

Zebrafish offer an excellent tool for studying the vertebrate hematopoietic system thanks to a highly conserved and rapidly developing hematopoietic program, genetic amenability, optical transparency, and experimental accessibility. Zebrafish studies have contributed to our understanding of hematopoiesis, a complex process regulated by signaling cues, inflammation being crucial among them. Hematopoietic stem cells (HSCs) are multipotent cells producing all the functional blood cells, including immune cells. HSCs respond to inflammation during infection and malignancy by proliferating and producing the blood cells in demand for a specific scenario. We first focus on how inflammation plays a crucial part in steady-state HSC development and describe the critical role of the inflammasome complex in regulating HSC expansion and balanced lineage production. Next, we review zebrafish studies of inflammatory innate immune mechanisms focusing on interferon signaling and the downstream JAK-STAT pathway. We also highlight insights gained from zebrafish models harbouring genetic perturbations in the role of inflammation in hematopoietic disorders such as bone marrow failure, myelodysplastic syndrome, and myeloid leukemia. Indeed, inflammation has been recently identified as a potential driver of clonal hematopoiesis and leukemogenesis, where cells acquire somatic mutations that provide a proliferative advantage in the presence of inflammation. Important insights in this area come from mutant zebrafish studies showing that hematopoietic differentiation can be compromised by epigenetic dysregulation and the aberrant induction of signaling pathways.

Stress hematopoiesis induces a proliferative advantage in TET2 deficiency.

TET2 loss-of-function mutations are recurrent events in a wide range of hematological malignancies and a physiologic occurrence in blood cells of healthy older adults. It is currently unknown what determines if a person harboring a somatic TET2 mutation will progress to myelodysplastic syndrome or acute myeloid leukemia. Here we develop a zebrafish tet2 mutant through which we show that tet2 loss leads to restricted hematopoietic differentiation combined with a modest upregulation of p53, which is also characteristic of many inherited bone marrow failure syndromes. Uniquely in the context of emergency hematopoiesis by external stimuli, such as infection or cytokine stimulation, lack of tet2 leads hematopoietic stem cells to undergo excessive proliferation, resulting in an accumulation of immature cells, which are poised to become leukemogenic following additional genetic/epigenetic perturbations. This same phenomenon observed in zebrafish extends to human hematopoietic stem cells, identifying TET2 as a critical relay switch in the context of stress hematopoiesis.

Zebrafish Cancer Predisposition Models.

Cancer predisposition syndromes are rare, typically monogenic disorders that result from germline mutations that increase the likelihood of developing cancer. Although these disorders are individually rare, resulting cancers collectively represent 5-10% of all malignancies. In addition to a greater incidence of cancer, affected individuals have an earlier tumor onset and are frequently subjected to long-term multi-modal cancer screening protocols for earlier detection and initiation of treatment. In vivo models are needed to better understand tumor-driving mechanisms, tailor patient screening approaches and develop targeted therapies to improve patient care and disease prognosis. The zebrafish (Danio rerio) has emerged as a robust model for cancer research due to its high fecundity, time- and cost-efficient genetic manipulation and real-time high-resolution imaging. Tumors developing in zebrafish cancer models are histologically and molecularly similar to their human counterparts, confirming the validity of these models. The zebrafish platform supports both large-scale random mutagenesis screens to identify potential candidate/modifier genes and recently optimized genome editing strategies. These techniques have greatly increased our ability to investigate the impact of certain mutations and how these lesions impact tumorigenesis and disease phenotype. These unique characteristics position the zebrafish as a powerful in vivo tool to model cancer predisposition syndromes and as such, several have already been created, including those recapitulating Li-Fraumeni syndrome, familial adenomatous polyposis, RASopathies, inherited bone marrow failure syndromes, and several other pathogenic mutations in cancer predisposition genes. In addition, the zebrafish platform supports medium- to high-throughput preclinical drug screening to identify compounds that may represent novel treatment paradigms or even prevent cancer evolution. This review will highlight and synthesize the findings from zebrafish cancer predisposition models created to date. We will discuss emerging trends in how these zebrafish cancer models can improve our understanding of the genetic mechanisms driving cancer predisposition and their potential to discover therapeutic and/or preventative compounds that change the natural history of disease for these vulnerable children, youth and adults.

Genome Editing in Zebrafish Using High-Fidelity Cas9 Nucleases: Choosing the Right Nuclease for the Task.

Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.

Frizzled 4 regulates ventral blood vessel remodeling in the zebrafish retina.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR. METHODS: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses. RESULTS: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination. CONCLUSION: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.

Zebrafish knock-ins swim into the mainstream.

The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study.

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Etiology and functional validation of gastrointestinal motility dysfunction in a zebrafish model of CHARGE syndrome.

CHARGE syndrome is linked to autosomal-dominant mutations in the CHD7 gene and results in a number of physiological and structural abnormalities, including heart defects, hearing and vision loss, and gastrointestinal (GI) problems. Of these challenges, GI problems have a profound impact throughout an individual's life, resulting in increased morbidity and mortality. A homolog of CHD7 has been identified in the zebrafish, the loss of which recapitulates many of the features of the human disease. Using a morpholino chd7 knockdown model complemented by a chd7 null mutant zebrafish line, we examined GI structure, innervation, and motility in larval zebrafish. Loss of chd7 resulted in physically smaller GI tracts with normal epithelial and muscular histology, but decreased and disorganized vagal projections, particularly in the foregut. chd7 morphant larvae had significantly less ability to empty their GI tract of gavaged fluorescent beads, and this condition was only minimally improved by the prokinetic agents, domperidone and erythromycin, in keeping with mixed responses to these agents in patients with CHARGE syndrome. The conserved genetics and transparency of the zebrafish have provided new insights into the consequences of chd7 gene dysfunction on the GI system and cranial nerve patterning. These findings highlight the opportunity of the zebrafish to serve as a preclinical model for studying compounds that may improve GI motility in individuals with CHARGE syndrome.

Cardiac Electrophysiological Effects of Light-Activated Chloride Channels.

During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl--driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.

hace1 Influences zebrafish cardiac development via ROS-dependent mechanisms.

BACKGROUND: In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes. RESULTS: We demonstrate that loss of hace1 in zebrafish via morpholino knockdown results in cardiac deformities, specifically a looping defect, where the heart is either tubular or "inverted". Whole-mount in situ hybridization of cardiac markers shows distinct abnormalities in ventricular morphology and atrioventricular valve formation in the hearts of these morphants, as well as increased expression of rac1. Importantly, this phenotype appears to be directly related to Nox enzyme-dependent ROS production, as both genetic inhibition by nox1 and nox2 morpholinos or pharmacologic rescue using ROS scavenging agents restores normal cardiac structure. CONCLUSIONS: Our study demonstrates that HACE1 is critical in the normal development and proper function of the vertebrate heart via a ROS-dependent mechanism. Developmental Dynamics 247:289-303, 2018. © 2017 Wiley Periodicals, Inc.

New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish.

INTRODUCTION: Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish. OBJECTIVE: To achieve temporal and spatial precision of genome editing, many new technologies can be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically- inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal control of gene regulation. In addition to controlling where and when genome editing occurs, using oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome. CONCLUSION: We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9.

The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.

Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements.

Out of the myriad of potential DNA binding sites of the glucocorticoid receptor (GR) found in the human genome, only a cell-type specific minority is actually bound, indicating that the presence of a recognition sequence alone is insufficient to specify where GR binds. Cooperative interactions with other transcription factors (TFs) are known to contribute to binding specificity. Here, we reasoned that sequence signals preventing GR recruitment to certain loci provide an alternative means to confer specificity. Motif analyses uncovered candidate Negative Regulatory Sequences (NRSs) that interfere with genomic GR binding. Subsequent functional analyses demonstrated that NRSs indeed prevent GR binding to nearby response elements. We show that NRS activity is conserved across species, found in most tissues and that they also interfere with the genomic binding of other TFs. Interestingly, the effects of NRSs appear not to be a simple consequence of changes in chromatin accessibility. Instead, we find that NRSs interact with proteins found at sub-nuclear structures called paraspeckles and that these proteins might mediate the repressive effects of NRSs. Together, our studies suggest that the joint influence of positive and negative sequence signals partition the genome into regions where GR can bind and those where it cannot.