Genomic selection signatures in autism spectrum disorder identifies cognitive genomic tradeoff and its relevance in paradoxical phenotypes of deficits versus potentialities.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by paradoxical phenotypes of deficits as well as gain in brain function. To address this a genomic tradeoff hypothesis was tested and followed up with the biological interaction and evolutionary significance of positively selected ASD risk genes. SFARI database was used to retrieve the ASD risk genes while for population datasets 1000 genome data was used. Common risk SNPs were subjected to machine learning as well as independent tests for selection, followed by Bayesian analysis to identify the cumulative effect of selection on risk SNPs. Functional implication of these positively selected risk SNPs was assessed and subjected to ontology analysis, pertaining to their interaction and enrichment of biological and cellular functions. This was followed by comparative analysis with the ancient genomes to identify their evolutionary patterns. Our results identified significant positive selection signals in 18 ASD risk SNPs. Functional and ontology analysis indicate the role of biological and cellular processes associated with various brain functions. The core of the biological interaction network constitutes genes for cognition and learning while genes in the periphery of the network had direct or indirect impact on brain function. Ancient genome analysis identified de novo and conserved evolutionary selection clusters. The de-novo evolutionary cluster represented genes involved in cognitive function. Relative enrichment of the ASD risk SNPs from the respective evolutionary cluster or biological interaction networks may help in addressing the phenotypic diversity in ASD. This cognitive genomic tradeoff signatures impacting the biological networks can explain the paradoxical phenotypes in ASD.

SCN9A Epileptic Encephalopathy Mutations Display a Gain-of-function Phenotype and Distinct Sensitivity to Oxcarbazepine.

Genetic mutants of voltage-gated sodium channels (VGSCs) are considered to be responsible for the increasing number of epilepsy syndromes. Previous research has indicated that mutations of one of the VGSC genes, SCN9A (Nav1.7), result in febrile seizures and Dravet syndrome in humans. Despite these recent efforts, the electrophysiological basis of SCN9A mutations remains unclear. Here, we performed a genetic screen of patients with febrile seizures and identified a novel missense mutation of SCN9A (W1150R). Electrophysiological characterization of different SCN9A mutants in HEK293T cells, the previously-reported N641Y and K655R variants, as well as the newly-found W1150R variant, revealed that the current density of the W1150R and N641Y variants was significantly larger than that of the wild-type (WT) channel. The time constants of recovery from fast inactivation of the N641Y and K655R variants were markedly lower than in the WT channel. The W1150R variant caused a negative shift of the G-V curve in the voltage dependence of steady-state activation. All mutants displayed persistent currents larger than the WT channel. In addition, we found that oxcarbazepine (OXC), one of the antiepileptic drugs targeting VGSCs, caused a significant shift to more negative potential for the activation and inactivation in WT and mutant channels. OXC-induced inhibition of currents was weaker in the W1150R variant than in the WT. Furthermore, with administering OXC the time constant of the N641Y variant was longer than those of the other two SCN9A mutants. In all, our results indicated that the point mutation W1150R resulted in a novel gain-of-function variant. These findings indicated that SCN9A mutants contribute to an increase in seizure, and show distinct sensitivity to OXC.

Calcineurin controls gene transcription following stimulation of a Gαq-coupled designer receptor.

Stimulation of Gaq-coupled receptors triggers the activation of gene transcription via a rise of intracellular Ca2+. To investigate the role of the Ca2+/calmodulin-dependent phosphatase calcineurin in regulating transcription following Gαq-coupled receptor stimulation, we used a gain-of-function approach and expressed ΔCnA, a constitutively active mutant of calcineurin A. Furthermore, we expressed hM3Dq, a designer receptor that is specifically coupled to Gαq and can be activated by the pharmacological compound clozapine-N-oxide. Stimulation of hM3Dq or expression of ΔCnA induced transcription of a reporter gene controlled by the calcineurin substrate nuclear factor of activated T cells (NFAT), suggesting that calcineurin increased NFAT-regulated gene transcription. In contrast, expression of ΔCnA attenuated hM3Dq-induced biosynthesis of the transcription factors c-Fos and Egr-1 and reduced both c-Fos and Egr-1 promoter activities. A dissection of the c-Fos and Egr-1 promoters revealed that calcineurin inhibited serum response element-mediated transcription. In particular, the expression of ΔCnA reduced the transcriptional activity of the ternary complex factor Elk-1 following stimulation of hM3Dq receptors. Furthermore, ΔCnA reduced the transcriptional activity of the transcription factor CREB and thus attenuated transcription mediated by the cAMP response element. In summary, we show that calcineurin functions as a positive and negative modulator of gene transcription.

Iron regulatory protein 2 is a suppressor of mutant p53 in tumorigenesis.

p53 is known to play a role in iron homeostasis and is required for FDXR-mediated iron metabolism via iron regulatory protein 2 (IRP2). Interestingly, p53 is frequently mutated in tumors wherein iron is often accumulated, suggesting that mutant p53 may exert its gain of function by altering iron metabolism. In this study, we found that FDXR deficiency decreased mutant p53 expression along with altered iron metabolism in p53R270H/- MEFs and cancer cells carrying mutant p53. Consistently, we found that decreased expression of mutant p53 by FDXR deficiency inhibited mutant p53-R270H to induce carcinoma and high grade pleomorphic sarcoma in FDXR+/-; p53R270H/- mice as compared with p53R270H/- mice. Moreover, we found that like its effect on wild-type p53, loss of IRP2 increased mutant p53 expression. However, unlike its effect to suppress cell growth in cells carrying wild-type p53, loss of IRP2 promoted cell growth in cancer cells expressing mutant p53. Finally, we found that ectopic expression of IRP2 suppressed cell growth in a mutant p53-dependent manner. Together, our data indicate that mutant p53 gain-of-function can be suppressed by IRP2 and FDXR deficiency, both of which may be explored to target tumors carrying mutant p53.

A DNMT3A PWWP mutation leads to methylation of bivalent chromatin and growth retardation in mice.

DNA methyltransferases (DNMTs) deposit DNA methylation, which regulates gene expression and is essential for mammalian development. Histone post-translational modifications modulate the recruitment and activity of DNMTs. The PWWP domains of DNMT3A and DNMT3B are posited to interact with histone 3 lysine 36 trimethylation (H3K36me3); however, the functionality of this interaction for DNMT3A remains untested in vivo. Here we present a mouse model carrying a D329A point mutation in the DNMT3A PWWP domain. The mutation causes dominant postnatal growth retardation. At the molecular level, it results in progressive DNA hypermethylation across domains marked by H3K27me3 and bivalent chromatin, and de-repression of developmental regulatory genes in adult hypothalamus. Evaluation of non-CpG methylation, a marker of de novo methylation, further demonstrates the altered recruitment and activity of DNMT3AD329A at bivalent domains. This work provides key molecular insights into the function of the DNMT3A-PWWP domain and role of DNMT3A in regulating postnatal growth.

Targeting AU-rich element-mediated mRNA decay with a truncated active form of the zinc-finger protein TIS11b/BRF1 impairs major hallmarks of mammary tumorigenesis.

Altered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnCS334D, by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnCS334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnCS334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnCS334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnCS334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnCS334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer.

Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia.

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D1 expressing MSNs of the direct pathway, as well as the D2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gαs-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.

Dysregulatory syndromes: the role of signal transducers and activators of transcription.

PURPOSE OF REVIEW: A comparative description of dysregulatory syndromes with mutations in signal transducer and activator of transcription (STAT) genes. RECENT FINDINGS: STAT 1, 3 and 5b loss of function (LOF) and gain of function (GOF) mutations are a heterogeneous group of genetic disorders that range from immunodeficiency (ID) to autoimmune disease (AID), depending on the underlying signalling pathway defect. Between them, there are clear overlapping and differences in clinical presentation and laboratory findings. SUMMARY: Dysregulatory syndromes due to LOF and GOF mutations in STAT1, 3 and 5b are a particular group of primary immunodeficiencies (PIDs) in which AID may be the predominant finding in addition to infections susceptibility. STAT1 GOF mutations were described as the major cause of chronic mucocutaneous candidiasis, while activating STAT3 mutations result in early-onset multiorgan autoimmunity and ID. Human STAT5b deficiency is a rare disease that also involves ID and severe growth failure. In recent years, the identification of the genes involved in these disorders allowed to differentiate these overlapping syndromes in order to choose the most effective therapeutic options.

ATXN1-CIC Complex Is the Primary Driver of Cerebellar Pathology in Spinocerebellar Ataxia Type 1 through a Gain-of-Function Mechanism.

Polyglutamine (polyQ) diseases are caused by expansion of translated CAG repeats in distinct genes leading to altered protein function. In spinocerebellar ataxia type 1 (SCA1), a gain of function of polyQ-expanded ataxin-1 (ATXN1) contributes to cerebellar pathology. The extent to which cerebellar toxicity depends on its cognate partner capicua (CIC), versus other interactors, remains unclear. It is also not established whether loss of the ATXN1-CIC complex in the cerebellum contributes to disease pathogenesis. In this study, we exclusively disrupt the ATXN1-CIC interaction in vivo and show that it is at the crux of cerebellar toxicity in SCA1. Importantly, loss of CIC in the cerebellum does not cause ataxia or Purkinje cell degeneration. Expression profiling of these gain- and loss-of-function models, coupled with data from iPSC-derived neurons from SCA1 patients, supports a mechanism in which gain of function of the ATXN1-CIC complex is the major driver of toxicity.

Activated ALK signals through the ERK-ETV5-RET pathway to drive neuroblastoma oncogenesis.

Activating mutations of the ALK receptor occur in a subset of neuroblastoma tumors. We previously demonstrated that Alk mutations cooperate with MYCN overexpression to induce neuroblastoma in mice and identified Ret as being strongly upregulated in MYCN/Alkmut tumors. By a genetic approach in vivo, we now document an oncogenic cooperation between activated Ret and MYCN overexpression in neuroblastoma formation. We show that MYCN/RetM919T tumors exhibit histological features and expression profiles close to MYCN/Alkmut tumors. We show that RET transcript levels decrease precedes RET protein levels decrease upon ALK inhibition in neuroblastoma cell lines. Etv5 was identified as a candidate transcription factor regulating Ret expression from murine MYCN/Alkmut tumor transcriptomic data. We demonstrate that ETV5 is regulated both at the protein and mRNA levels upon ALK activation or inhibition in neuroblastoma cell lines and that this regulation precedes RET modulation. We document that ALK activation induces ETV5 protein upregulation through stabilization in a MEK/ERK-dependent manner. We show that RNAi-mediated inhibition of ETV5 decreases RET expression. Reporter assays indicate that ETV5 is able to drive RET gene transcription. ChIP-seq analysis confirmed ETV5 binding on the RET promoter and identified an enhancer upstream of the promoter. Finally, we demonstrate that combining RET and ALK inhibitors reduces tumor growth more efficiently than each single agent in MYCN and AlkF1178L-driven murine neuroblastoma. Altogether, these results define the ERK-ETV5-RET pathway as a critical axis driving neuroblastoma oncogenesis downstream of activated ALK.

Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cells by targeting the transcription factors FOXO3a and FOXM1.

Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that promote cancer cell invasive growth and metastasis, yet the mechanisms mediating these functions still largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances the invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in in vitro three-dimensional culture and in an in vivo orthotopic nude mouse model of HNSCC through a novel transcription-independent mechanism. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the AMP-activated protein kinase (AMPK)-mediated phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. This downregulation of FOXO3a's activity, in turn, leads to de-repression of FOXM1 expression. Importantly, we show that either overexpression of FOXO3a or downregulation of FOXM1 impairs both GOF mutant p53-mediated cell invasion in vitro and pulmonary metastases of UM-SCC-1 cells in vivo. Finally, not only do oral cancer patients with p53 mutations exhibit higher levels of FOXM1 expression than patients with wild-type p53, but also HNSCC patients with TP53 mutations and high levels of FOXM1 expression have the poorest survival outcomes. Given our prior demonstration that GOF mutant p53s inhibit AMPK, our current study, establishes and demonstrates a novel transcription-independent GOF mutant p53-AMPK-FOXO3a-FOXM1 signaling cascade that plays an important role in mediating mutant p53s' gain-of-function activities in HNSCCs.

Heterogeneity in the Drosophila gustatory receptor complexes that detect aversive compounds.

Animals must detect aversive compounds to survive. Bitter taste neurons express heterogeneous combinations of bitter receptors that diversify their response profiles, but this remains poorly understood. Here we describe groups of taste neurons in Drosophila that detect the same bitter compounds using unique combinations of gustatory receptors (GRs). These distinct complexes also confer responsiveness to non-overlapping sets of additional compounds. While either GR32a/GR59c/GR66a or GR22e/GR32a/GR66a heteromultimers are sufficient for lobeline, berberine, and denatonium detection, only GR22e/GR32a/GR66a responds to strychnine. Thus, despite minimal sequence-similarity, Gr22e and Gr59c show considerable but incomplete functional overlap. Since the gain- or loss-of-function of Gr22e or Gr59c alters bitter taste response profiles, we conclude a taste neuron's specific combination of Grs determines its response profile. We suspect the heterogeneity of Gr expression in Drosophila taste neurons diversifies bitter compound detection, improving animal fitness under changing environmental conditions that present a variety of aversive compounds.