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Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in ‘regulation of RNA splicing’ and ‘mRNA catabolic process’. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouseAS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)—containing the invSINEB2 element—able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.

Alternative splicing (AS) appears to be altered in Huntington’s disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected.Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a strong correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, expanded Htt CAG repeats reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors.Though the mechanisms of this dysregulation remain uncertain, our study unveils network of transcriptionally altered micro-RNAs and RNA-binding proteins (CELF, hnRNPS, PTBP, SRSF) which, in turn, might influence the AS machinery, primarily in neural cells.We suggest that this unbalanced expression of linear and circular RNAs might result in altered neural fitness, contributing to HD striatal vulnerability.

Background With the development of new therapies entering clinical trials for HD, there is an increasing need to develop and validate biomarkers in accessible biofluids, such as blood, to follow disease progression and predict treatment outcomes. Some biomarkers do exist; however, reliable and readily available additional ones will be crucial in assessing the pathogenic process and pharmacologic responses to therapeutic interventions. Aims For the first time in HD, we detect and characterize circRNAs in peripheral blood of a cohort of gender and age-matched controls and HD patients at various stages of the disease. We studied their potential as biomarkers by evaluating their correlation with disease progression and the size of the CAG repeat. Methods 50 blood samples were collected from both HD patients and a healthy cohort of individuals. Total RNA was used for RNA sequencing, and the obtained data were aligned with STAR. DCC program was used to quantify circRNAs and CircTest (R package) to identify differentially expressed circRNA between different disease stages and controls. CAG repeat size correlation was also tested. To validate our findings, candidate circRNAs were quantified via RT-qPCR in our samples. Results Our study led to the discovery of 7, stringently defined circRNAs that possess 3 characteristics: differentially expressed in HD patients (all up-regulated, p-value 0.3 and p-value Conclusions We identified for the first time circRNAs whose expression increases in the blood of HD patients. Currently, we aim to validate these circular molecules in independent blood cohorts as well as in cerebrospinal fluid and plasma samples to fully elucidate whether these highly stable RNA molecules could be used as disease biomarkers.

Background Circular RNAs (circRNAs) are a special group of non-coding RNAs formed by back-splicing. Mostly cytosolic in eukaryotic cells, circRNAs are particularly enriched and conserved in neurons and originate from protein-coding genes to function as global regulators of gene expression. Recent studies showed that circRNAs partake in brain physiology and pathology, contributing to neurological disorders, such as myotonic dystrophy type 1, Parkinson’s and Alzheimer’s Diseases. Here, we identified the first ever known brain enriched RNA circle originating from the Huntington’s Disease gene HTT (CircHTT: 484 nt, Ex 2-6, chr4:3088665-3109150) which is conserved in mouse and minipig. Aims and Methods To functionally characterize circHTT and its implication for HD pathogenesis, we first studied its expression pattern in human and mouse body districts and in iPS-derived neuronal cell lines with different CAG repeats. Then, overexpression and down-regulation of the circle were used to determine the effects on transcription of the HD gene and translation of wild-type and mutant protein. Results CircHTT expression increases significantly with increasing CAG repeats in terminally differentiated cortical neurons. Furthermore, circHtt is significantly more expressed in brain districts of Q111 and zQ175 knock-in mice. These findings indicate that the expression of circHTT/Htt occurs in a CAG repeat dependent manner in neuronal cells, typical hallmark of HD pathology. Strikingly, overexpression of the circular RNA in human HEK293T, PC3, mouse STHdh Q7/7, Q7/111 and Q111/111 cell lines consistently increase wild-type huntingtin, while decreasing mutant huntingtin protein, with no alteration of the HTT/Htt transcript level. Conclusions In conclusion, we identified a brain enriched RNA circle originating from the HD gene locus that is sensitive to the HD mutation and may modulate huntingtin expression. Our observations might pave the way to new trials of therapeutic intervention.

Huntington’s disease (HD) is a devastating neurodegenerative disorder caused by an aberrant expansion of the CAG tract within the exon 1 of the HD gene, HTT. HD progressively impairs motor and cognitive capabilities, leading to a total loss of autonomy and ultimate death. Currently, no cure or effective treatment is available to halt the disease. Although the HTT gene is ubiquitously expressed, the striatum appears to be the most susceptible district to the HD mutation with Medium-sized Spiny Neurons (MSNs) (D1R and D2R) representing 95% of the striatal neuronal population. Why are striatal MSNs so vulnerable to the HD mutation? Particularly, why do D1R- and D2R-MSNs display different susceptibility to HD? Here, we highlight significant differences between D1R- and D2R-MSNs subpopulations, such as morphology, electrophysiology, transcriptomic, functionality, and localization in the striatum. We discuss possible reasons for their selective degeneration in the context of HD. Our review suggests that a better understanding of cell type-specific gene expression dysregulation within the striatum might reveal new paths to therapeutic intervention or prevention to ameliorate HD patients’ life expectancy.

Disruptive mutations in the chromodomain helicase DNA binding protein 8 (CHD8) have been recurrently associated with Autism Spectrum Disorders (ASD). In normal cellular physiology, CHD8 co-purifies with MLL1 and MOF transcriptional activation complex, with elongating RNAPII and directly binds to DNA promoters and enhancers regions, thus a regulatory role in transcriptional initiation and elongation could be postulated.Here we investigated how chromatin landscape reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. We interrogated transcriptionally active and repressed regions, as well as active and poised enhancers.CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes. Histone H3K36me3 reduction associated to CHD8-suppression does not functionally impact on global transcriptional levels, but correlated with altered alternative splicing patterns of ∼ 2000 protein coding genes implicated in “RNA splicing”, “mitotic cell cycle phase transition” and “mRNA processing”, especially affecting alternative first exon and exon skipping events.In summary, our results point toward broad molecular consequences of CHD8 suppression, implicating altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

Background Huntington’s Disease (HD), is a hereditary, fatal neurodegenerative disorder due to CAG trinucleotide expansion in exon 1 of the HD gene (HTT). Recent findings revealed that the process of alternative splicing (AS) might be compromised in HD. AS regulation is crucial not only to the establishment of a repertoire of protein coding isoforms extremely relevant for the proper physiological characteristics of the nervous system, but also to the biogenesis of circular RNAs (circRNAs), unusually stable, highly expressed non-coding RNAs produced by the circularization of exons which, seems to have important roles during neuronal development and functioning. Aims Therefore, the overarching goal of our research was to take advantage of genetically engineered Htt knock-in (KI) mouse cellular and in vivo model systems bearing normal or pathological Htt CAG repeat lengths to discover alterations in linear and back-splicing events that might reflect on the levels of expression as well as the composition of a repertoire of proteins, thus contributing to HD striatal vulnerability and pathogenesis. Methods/techniques To discover alterations in linear splicing, we resourced to a publicly available dataset (Langfelder P. et al., 2016, Nature Neuroscience) analyzing full transcriptomic profiling dataset for striatum, cortex and liver at different developmental time points, identifying and quantifying the total numbers of differential AS events for each genotype/sample type and time point. Results/outcome Our analysis demonstrated that specifically for the striatum – most vulnerable to HD degeneration – the total number of detected, differential AS events increased significantly at 6 and 10 month with increasing CAG expansion. Interestingly, not all the AS events increased with CAG-expansion and age, but only a specific subtype of AS event – EXON SKIPPING – revealed to be strongly and specifically affected. On the other hand, we identified and characterized the first circular RNA originating locally from the HTT locus, expressed in whole body, but predominantly in the brain and spinal cord and presenting augmented expression level with increasing HTT CAG size. Moreover, at genome-wide level, data analysis of circRNA-seq showed a decreased circRNAs production when mutant huntingtin is expressed. Specifically, 12 circRNAs, identified by stringent cut-off criteria, showed continuous decreased expression following CAG expansion in neuronal progenitor cells. Since, most of the CAG-sensitive circRNAs have annotated human orthologues, their expression can be further characterized and functionally studied in human cell lines. Conclusions In conclusion, our results support the idea that AS machinery is responding to HD mutational process altering both linear and back-splicing events locally at the HTT locus, but also at genome-wide level. This knowledge will pave the way to new trials of therapeutic intervention aimed to possibly target spliceosomal-circRNAs alterations through specific drugs or genetic manipulation.

Huntington's disease (HD) is a genetic, fatal autosomal dominant neurodegenerative disorder typically occurring in midlife with symptoms ranging from chorea, to dementia, to personality disturbances (Philos Trans R Soc Lond Ser B Biol Sci 354:957-961, 1999). HD is inherited in a dominant fashion, and the underlying mutation in all cases is a CAG trinucleotide repeat expansion within exon 1 of the HD gene (Cell 72:971-983, 1993). The expanded CAG repeat, translated into a lengthened glutamine tract at the amino terminus of the huntingtin protein, affects its structural properties and functional activities. The effects are pleiotropic, as huntingtin is broadly expressed in different cellular compartments (i.e., cytosol, nucleus, mitochondria) as well as in all cell types of the body at all developmental stages, such that HD pathogenesis likely starts at conception and is a lifelong process (Front Neurosci 9:509, 2015). The rate-limiting mechanism(s) of neurodegeneration in HD still remains elusive: many different processes are commonly disrupted in HD cell lines and animal models, as well as in HD patient cells (Eur J Neurosci 27:2803-2820, 2008); however, epigenetic-chromatin deregulation, as determined by the analysis of DNA methylation, histone modifications, and noncoding RNAs, has now become a prevailing feature. Thus, the overarching goal of this chapter is to discuss the current status of the literature, reviewing how an aberrant epigenetic landscape can contribute to altered gene expression and neuronal dysfunction in HD.

Huntington's disease (HD) is a genetic, fatal autosomal dominant neurodegenerative disorder typically occurring in midlife with symptoms ranging from chorea, to dementia, to personality disturbances (Philos Trans R Soc Lond Ser B Biol Sci 354:957-961, 1999). HD is inherited in a dominant fashion, and the underlying mutation in all cases is a CAG trinucleotide repeat expansion within exon 1 of the HD gene (Cell 72:971-983, 1993). The expanded CAG repeat, translated into a lengthened glutamine tract at the amino terminus of the huntingtin protein, affects its structural properties and functional activities. The effects are pleiotropic, as huntingtin is broadly expressed in different cellular compartments (i.e., cytosol, nucleus, mitochondria) as well as in all cell types of the body at all developmental stages, such that HD pathogenesis likely starts at conception and is a lifelong process (Front Neurosci 9:509, 2015). The rate-limiting mechanism(s) of neurodegeneration in HD still remains elusive: many different processes are commonly disrupted in HD cell lines and animal models, as well as in HD patient cells (Eur J Neurosci 27:2803-2820, 2008); however, epigenetic-chromatin deregulation, as determined by the analysis of DNA methylation, histone modifications, and noncoding RNAs, has now become a prevailing feature. Thus, the overarching goal of this chapter is to discuss the current status of the literature, reviewing how an aberrant epigenetic landscape can contribute to altered gene expression and neuronal dysfunction in HD.

Huntington's disease (HD) is a monogenic autosomal dominant, fatal disorder due to CAG trinucleotide expansion in exon 1 of the HD gene (HTT) (The Huntington's Disease Collaborative Research Group, 1993; Figure ​Figure1A).1A). Nowadays, there is no cure or effective treatment for the disease which presents with motor, cognitive and psychiatric dysfunction. Although typically conceived as a “neurodegenerative disease,” mainly affecting the GABAergic medium-sized spiny neurons (MSN) of the striatum and deep layers of the cortex (Rosas et al., 2003), an increasing body of evidence has surfaced indicating that abnormal neurodevelopment might also have a crucial role in HD (Mehler and Gokhan, 2000, 2001; Humbert, 2010). Neurodegenerative diseases have been classically defined as chronic and progressive disorders of the nervous system affecting neurologic and behavioral functions, which start with specific biochemical changes that ultimately lead to distinct histopathologic and clinical syndromes. On the contrary, neurodevelopmental disorders result from an anomaly of brain maturation, during fetal or early postnatal life, which is postulated to alter the structure and/or function of neuronal and synaptic populations (Harrison, 1995).

Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover from the toxin attack. The fact that the majority of the human transcriptome encodes for non-coding RNAs (ncRNAs) raises the question: do host cells deploy non-coding transcripts to rapidly control the most energy-consuming process in cells--i.e., host translation--to counteract the infection? Here, we discuss the intriguing possibility that membrane-damaging toxins induce, in the host, the expression of toxin-specific long non-coding RNAs (lncRNAs), which act as sponges for other molecules, encoding small peptides or binding target mRNAs to depress their translation efficiency. Unravelling the function of host-produced lncRNAs upon bacterial infection or membrane damage requires an improved understanding of host lncRNA expression patterns, their association with polysomes and their function during this stress. This field of investigation holds a unique opportunity to reveal unpredicted scenarios and novel approaches to counteract antibiotic-resistant infections.

The CAG repeat expansion in the Huntington's disease gene HTT extends a polyglutamine tract in mutant huntingtin that enhances its ability to facilitate polycomb repressive complex 2 (PRC2). To gain insight into this dominant gain of function, we mapped histone modifications genome-wide across an isogenic panel of mouse embryonic stem cell (ESC) and neuronal progenitor cell (NPC) lines, comparing the effects of Htt null and different size Htt CAG mutations. We found that Htt is required in ESC for the proper deposition of histone H3K27me3 at a subset of ‘bivalent’ loci but in NPC it is needed at ‘bivalent’ loci for both the proper maintenance and the appropriate removal of this mark. In contrast, Htt CAG size, though changing histone H3K27me3, is prominently associated with altered histone H3K4me3 at ‘active’ loci. The sets of ESC and NPC genes with altered histone marks delineated by the lack of huntingtin or the presence of mutant huntingtin, though distinct, are enriched in similar pathways with apoptosis specifically highlighted for the CAG mutation. Thus, the manner by which huntingtin function facilitates PRC2 may afford mutant huntingtin with multiple opportunities to impinge upon the broader machinery that orchestrates developmentally appropriate chromatin status.

In patients with Huntington's disease (HD), the protein huntingtin (Htt) has an expanded polyglutamine (poly-Q) tract. HD results in early loss of medium spiny neurons in the striatum, which impairs motor and cognitive functions. Identifying the physiological role and molecular functions of Htt may yield insight into HD pathogenesis. We found that Htt promotes signaling by mTORC1 [mechanistic target of rapamycin (mTOR) complex 1] and that this signaling is potentiated by poly-Q-expanded Htt. Knocking out Htt in mouse embryonic stem cells or human embryonic kidney cells attenuated amino acid-induced mTORC1 activity, whereas overexpressing wild-type or poly-Q-expanded Htt in striatal neuronal cells increased basal mTOR activity. Striatal cells expressing endogenous poly-Q-expanded Htt showed an increase in the number and size of mTOR puncta on the perinuclear regions compared to cells expressing wild-type Htt. Pull-down experiments indicated that amino acids stimulated the interaction of Htt and the guanosine triphosphatase (GTPase) Rheb (a protein that stimulates mTOR activity), and that Htt forms a ternary complex with Rheb and mTOR. Pharmacologically inhibiting PI3K (phosphatidylinositol 3-kinase) or knocking down Rheb abrogated mTORC1 activity induced by expression of a poly-Q-expanded amino-terminal Htt fragment. Moreover, striatum-specific deletion of TSC1, encoding tuberous sclerosis 1, a negative regulator of mTORC1, accelerated the onset of motor coordination abnormalities and caused premature death in an HD mouse model. Together, our findings demonstrate that mutant Htt contributes to the pathogenesis of HD by enhancing mTORC1 activity.

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10−8) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10−10). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.