Polygenic risk for schizophrenia converges on alternative polyadenylation as molecular mechanism underlying synaptic impairment.

Schizophrenia (SCZ) is a genetically heterogenous psychiatric disorder of highly polygenic nature. Correlative evidence from genetic studies indicate that the aggregated effects of distinct genetic risk factor combinations found in each patient converge onto common molecular mechanisms. To prove this on a functional level, we employed a reductionistic cellular model system for polygenic risk by differentiating induced pluripotent stem cells (iPSCs) from 104 individuals with high polygenic risk load and controls into cortical glutamatergic neurons (iNs). Multi-omics profiling identified widespread differences in alternative polyadenylation (APA) in the 3' untranslated region of many synaptic transcripts between iNs from SCZ patients and healthy donors. On the cellular level, 3'APA was associated with a reduction in synaptic density of iNs. Importantly, differential APA was largely conserved between postmortem human prefrontal cortex from SCZ patients and healthy donors, and strongly enriched for transcripts related to synapse biology. 3'APA was highly correlated with SCZ polygenic risk and affected genes were significantly enriched for SCZ associated common genetic variation. Integrative functional genomic analysis identified the RNA binding protein and SCZ GWAS risk gene PTBP2 as a critical trans-acting factor mediating 3'APA of synaptic genes in SCZ subjects. Functional characterization of PTBP2 in iNs confirmed its key role in 3'APA of synaptic transcripts and regulation of synapse density. Jointly, our findings show that the aggregated effects of polygenic risk converge on 3'APA as one common molecular mechanism that underlies synaptic impairments in SCZ.

Massively parallel functional dissection of schizophrenia-associated noncoding genetic variants.

Schizophrenia (SCZ) is a highly heritable mental disorder with thousands of associated genetic variants located mostly in the noncoding space of the genome. Translating these associations into insights regarding the underlying pathomechanisms has been challenging because the causal variants, their mechanisms of action, and their target genes remain largely unknown. We implemented a massively parallel variant annotation pipeline (MVAP) to perform SCZ variant-to-function mapping at scale in disease-relevant neural cell types. This approach identified 620 functional variants (1.7%) that operate in a highly developmental context and neuronal-activity-dependent manner. Multimodal integration of epigenomic and CRISPRi screening data enabled us to link these functional variants to target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a multistage prioritization strategy to map functional single-nucleotide polymorphism (SNP)-to-gene-to-endophenotype relations and offer biological insights into the context-dependent molecular processes modulated by SCZ-associated genetic variation.

Cell type and condition specific functional annotation of schizophrenia associated non-coding genetic variants.

Schizophrenia (SCZ) is a highly polygenic disease and genome wide association studies have identified thousands of genetic variants that are statistically associated with this psychiatric disorder. However, our ability to translate these associations into insights on the disease mechanisms has been challenging since the causal genetic variants, their molecular function and their target genes remain largely unknown. In order to address these questions, we established a functional genomics pipeline in combination with induced pluripotent stem cell technology to functionally characterize ~35,000 non-coding genetic variants associated with schizophrenia along with their target genes. This analysis identified a set of 620 (1.7%) single nucleotide polymorphisms as functional on a molecular level in a highly cell type and condition specific fashion. These results provide a high-resolution map of functional variant-gene combinations and offer comprehensive biological insights into the developmental context and stimulation dependent molecular processes modulated by SCZ associated genetic variation.

Associations of psychiatric disease and ageing with FKBP5 expression converge on superficial layer neurons of the neocortex.

Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry. FKBP5 is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported that FKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propel FKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n = 1024) of FKBP5/1, examining neocortical areas (BA9, BA11 and ventral BA24/BA24a) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease state, ageing and genotype on cortical FKBP5/1 expression including in a cell type-specific manner. We identified consistently heightened FKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq; BA9 and BA11) and targeted histology (BA9, BA24a), we established that these disease and ageing effects on FKBP5/1 expression were most pronounced in excitatory superficial layer neurons of the neocortex, and this effect appeared to be consistent in both the granular and agranular areas examined. We then found that this increase in FKBP5 levels may impact on synaptic plasticity, as FKBP5 gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in superficial layer neurons in BA11. These findings pinpoint a novel cellular and molecular mechanism that has potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.

Tripartite extended amygdala-basal ganglia CRH circuit drives locomotor activation and avoidance behavior.

An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional, and behavioral adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL), a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits hyperactivation of the hypothalamic-pituitary-adrenal axis, locomotor activation, and avoidance behavior contingent on CRH receptor type 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behavior is shaped by IPACLCRH and GPeCRHR1 neurons coalescing in the SN. These results delineate a previously unidentified tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive locomotor activation and avoidance behavior.

Persistent epigenetic memory impedes rescue of the telomeric phenotype in human ICF iPSCs following DNMT3B correction.

DNA methyltransferase 3B (DNMT3B) is the major DNMT that methylates mammalian genomes during early development. Mutations in human DNMT3B disrupt genome-wide DNA methylation patterns and result in ICF syndrome type 1 (ICF1). To study whether normal DNA methylation patterns may be restored in ICF1 cells, we corrected DNMT3B mutations in induced pluripotent stem cells from ICF1 patients. Focusing on repetitive regions, we show that in contrast to pericentromeric repeats, which reacquire normal methylation, the majority of subtelomeres acquire only partial DNA methylation and, accordingly, the ICF1 telomeric phenotype persists. Subtelomeres resistant to de novo methylation were characterized by abnormally high H3K4 trimethylation (H3K4me3), and short-term reduction of H3K4me3 by pharmacological intervention partially restored subtelomeric DNA methylation. These findings demonstrate that the abnormal epigenetic landscape established in ICF1 cells restricts the recruitment of DNMT3B, and suggest that rescue of epigenetic diseases with genome-wide disruptions will demand further manipulation beyond mutation correction.

HiCeekR: A Novel Shiny App for Hi-C Data Analysis.

The High-throughput Chromosome Conformation Capture (Hi-C) technique combines the power of the Next Generation Sequencing technologies with chromosome conformation capture approach to study the 3D chromatin organization at the genome-wide scale. Although such a technique is quite recent, many tools are already available for pre-processing and analyzing Hi-C data, allowing to identify chromatin loops, topological associating domains and A/B compartments. However, only a few of them provide an exhaustive analysis pipeline or allow to easily integrate and visualize other omic layers. Moreover, most of the available tools are designed for expert users, who have great confidence with command-line applications. In this paper, we present HiCeekR (https://github.com/lucidif/HiCeekR), a novel R Graphical User Interface (GUI) that allows researchers to easily perform a complete Hi-C data analysis. With the aid of the Shiny libraries, it integrates several R/Bioconductor packages for Hi-C data analysis and visualization, guiding the user during the entire process. Here, we describe its architecture and functionalities, then illustrate its capabilities using a publicly available dataset.

Collagen Prolyl Hydroxylation-Dependent Metabolic Perturbation Governs Epigenetic Remodeling and Mesenchymal Transition in Pluripotent and Cancer Cells.

Collagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires vitamin C (VitC). Here, we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control CST and paves the way for the development of novel antimetastatic strategies. SIGNIFICANCE: A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3235/F1.large.jpg.

DNMT3B Functions: Novel Insights From Human Disease.

DNA methylation plays important roles in gene expression regulation and chromatin structure. Its proper establishment and maintenance are essential for mammalian development and cellular differentiation. DNMT3B is the major de novo DNA methyltransferase expressed and active during the early stage of embryonic development, including implantation. In addition to its well-known role to methylate centromeric, pericentromeric, and subtelomeric repeats, recent observations suggest that DNMT3B acts as the main enzyme methylating intragenic regions of active genes. Although largely studied, much remains unknown regarding how these specific patterns of de novo CpG methylation are established in mammalian cells, and which are the rules governing DNMT3B recruitment and activity. Latest evidence indicates that DNMT3B recruitment is regulated by numerous mechanisms including chromatin modifications, transcription levels, non-coding RNAs, and the presence of DNA-binding factors. DNA methylation abnormalities are a common mark of human diseases involving chromosomal and genomic instabilities, such as inherited disease and cancer. The autosomal recessive Immunodeficiency, Centromeric instability and Facial anomalies syndrome, type I (ICF-1), is associated to hypomorphic mutations in DNMT3B gene, while its altered expression has been correlated with the development of tumors. In both cases, this implies that abnormal DNA hypomethylation and hypermethylation patterns affect gene expression and genomic architecture contributing to the pathological states. We will provide an overview of the most recent research aimed at deciphering the molecular mechanisms by which DNMT3B abnormalities are associated with the onset and progression of these pathologies.

Sphingosine Kinases promote IL-17 expression in human T lymphocytes.

Sphingosine 1-phosphate (S1P) has a role in many cellular processes. S1P is involved in cell growth and apoptosis, regulation of cell trafficking, production of cytokines and chemokines. The kinases SphK1 and SphK2 (SphKs) phosphorilate Sphingosine (Sph) to S1P and several phosphatases revert S1P to sphingosine, thus assuring a balanced pool that can be depleted by a Sphingosine lyase in hexadecenal compounds and aldehydes. There are evidences that SphK1 and 2 may per se control cellular processes. Here, we report that Sph kinases regulate IL-17 expression in human T cells. SphKs inhibition impairs the production of IL-17, while their overexpression up-regulates expression of the cytokine through acetylation of IL-17 promoter. SphKs were up-regulated also in PBMCs of patients affected by IL-17 related diseases. Thus, S1P/S1P kinases axis is a mechanism likely to promote IL-17 expression in human T cells, representing a possible therapeutic target in human inflammatory diseases.

An Ultraconserved Element Containing lncRNA Preserves Transcriptional Dynamics and Maintains ESC Self-Renewal.

Ultraconserved elements (UCEs) show the peculiar feature to retain extended perfect sequence identity among human, mouse, and rat genomes. Most of them are transcribed and represent a new family of long non-coding RNAs (lncRNAs), the transcribed UCEs (T-UCEs). Despite their involvement in human cancer, the physiological role of T-UCEs is still unknown. Here, we identify a lncRNA containing the uc.170+, named T-UCstem1, and provide in vitro and in vivo evidence that it plays essential roles in embryonic stem cells (ESCs) by modulating cytoplasmic miRNA levels and preserving transcriptional dynamics. Specifically, while T-UCstem1::miR-9 cytoplasmic interplay regulates ESC proliferation by reducing miR-9 levels, nuclear T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing polycomb repressive complex 2 on bivalent domains. Altogether, our findings provide unprecedented evidence that T-UCEs regulate physiological cellular functions and point to an essential role of T-UCstem1 in preserving ESC identity.

ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing.

Hypomorphic mutations in DNA-methyltransferase DNMT3B cause majority of the rare disorder Immunodeficiency, Centromere instability and Facial anomalies syndrome cases (ICF1). By unspecified mechanisms, mutant-DNMT3B interferes with lymphoid-specific pathways resulting in immune response defects. Interestingly, recent findings report that DNMT3B shapes intragenic CpG-methylation of highly-transcribed genes. However, how the DNMT3B-dependent epigenetic network modulates transcription and whether ICF1-specific mutations impair this process remains unknown. We performed a transcriptomic and epigenomic study in patient-derived B-cell lines to investigate the genome-scale effects of DNMT3B dysfunction. We highlighted that altered intragenic CpG-methylation impairs multiple aspects of transcriptional regulation, like alternative TSS usage, antisense transcription and exon splicing. These defects preferentially associate with changes of intragenic H3K4me3 and at lesser extent of H3K27me3 and H3K36me3. In addition, we highlighted a novel DNMT3B activity in modulating the self-regulatory circuit of sense-antisense pairs and the exon skipping during alternative splicing, through interacting with RNA molecules. Strikingly, altered transcription affects disease relevant genes, as for instance the memory-B cell marker CD27 and PTPRC genes, providing us with biological insights into the ICF1-syndrome pathogenesis. Our genome-scale approach sheds light on the mechanisms still poorly understood of the intragenic function of DNMT3B and DNA methylation in gene expression regulation.

RIP: RNA Immunoprecipitation.

The relevance of RNA-protein interactions in modulating mRNA and noncoding RNA function is increasingly appreciated and several methods have been recently developed to map them. The RNA immunoprecipitation (RIP) is a powerful method to study the physical association between individual proteins and RNA molecules in vivo. The basic principles of RIP are very similar to those of chromatin immunoprecipitation (ChIP), a largely used tool in the epigenetic field, but with some important caveats. The approach is based on the use of a specific antibody raised against the protein of interest to pull down the RNA-binding protein (RBP) and target-RNA complexes. Any RNA that is associated with this protein complex will also be isolated and can be further analyzed by polymerase chain reaction-based methods, hybridization, or sequencing.Several variants of this technique exist and can be divided into two main classes: native and cross-linked RNA immunoprecipitation. The native RIP allows to reveal the identity of RNAs directly bound by the protein and their abundance in the immunoprecipitated sample, while cross-linked RIP leads to precisely map the direct and indirect binding site of the RBP of interest to the RNA molecule.In this chapter both the protocols applied to mammalian cells are described taking into account the caveats and considerations required for designing, performing, and interpreting the results of these experiments.

Non-coding RNAs in chromatin disease involving neurological defects.

Novel classes of small and long non-coding RNAs (ncRNAs) are increasingly becoming apparent, being engaged in diverse structural, functional and regulatory activities. They take part in target gene silencing, play roles in transcriptional, post-transcriptional and epigenetic processes, such as chromatin remodeling, nuclear reorganization with the formation of silent compartments and fine-tuning of gene recruitment into them. Among their functions, non-coding RNAs are thought to act either as guide or scaffold for epigenetic modifiers that write, erase, and read the epigenetic signature over the genome. Studies on human disorders caused by defects in epigenetic modifiers and involving neurological phenotypes highlight the disruption of diverse classes of non-coding RNAs. Noteworthy, these molecules mediate a wide spectrum of neuronal functions, including brain development, and synaptic plasticity. These findings imply a significant contribution of ncRNAs in pathophysiology of the aforesaid diseases and provide new concepts for potential therapeutic applications.

L-Proline induces a mesenchymal-like invasive program in embryonic stem cells by remodeling H3K9 and H3K36 methylation.

Metabolites are emerging as key mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. We found that the nonessential amino acid L-proline (L-Pro) acts as a signaling molecule that promotes the conversion of embryonic stem cells into mesenchymal-like, spindle-shaped, highly motile, invasive pluripotent stem cells. This embryonic-stem-cell-to-mesenchymal-like transition (esMT) is accompanied by a genome-wide remodeling of the H3K9 and H3K36 methylation status. Consistently, L-Pro-induced esMT is fully reversible either after L-Pro withdrawal or by addition of ascorbic acid (vitamin C), which in turn reduces H3K9 and H3K36 methylation, promoting a mesenchymal-like-to-embryonic-stem-cell transition (MesT). These findings suggest that L-Pro, which is produced by proteolytic remodeling of the extracellular matrix, may act as a microenvironmental cue to control stem cell behavior.