Marco Magistri

Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation

The ability to specifically upregulate genes in vivo holds great therapeutic promise. Here we show that inhibition or degradation of natural antisense transcripts (NATs) by single-stranded oligonucleotides or siRNAs can transiently and reversibly upregulate locus-specific gene expression. Brain-derived neurotrophic factor (BDNF) is normally repressed by a conserved noncoding antisense RNA transcript, BDNF-AS. Inhibition of this transcript upregulates BDNF mRNA by two- to sevenfold, alters chromatin marks at the BDNF locus, leads to increased protein levels and induces neuronal outgrowth and differentiation both in vitro and in vivo. We also show that inhibition of NATs leads to increases in glial-derived neurotrophic factor (GDNF) and ephrin receptor B2 (EPHB2) mRNA. Our data suggest that pharmacological approaches targeting NATs can confer locus-specific gene upregulation effects.Here we demonstrate that natural antisense transcripts (NATs), which are abundant in mammalian genomes, can function as repressors of specific genomic loci and that their removal or inhibition by AntagoNAT oligonucleotides leads to transient and reversible upregulation of sense gene expression. As one example, we show that Brain-Derived Neurotrophic Factor (BDNF) is under the control of a conserved noncoding antisense RNA transcript, BDNF-AS, both in vitro and in vivo. BDNF-AS tonically represses BDNF sense RNA transcription by altering chromatin structure at the BDNF locus, which in turn reduces endogenous BDNF protein and function. By providing additional and analogous examples of endogenous mRNA upregulation, we suggest that antisense RNA mediated transcriptional suppression is a common phenomenon. In sum, we demonstrate a novel pharmacological strategy by which endogenous gene expression can be upregulated in a locus-specific manner.

Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts

In the decade following the publication of the Human Genome, noncoding RNAs (ncRNAs) have reshaped our understanding of the broad landscape of genome regulation. During this period, natural antisense transcripts (NATs), which are transcribed from the opposite strand of either protein or non-protein coding genes, have vaulted to prominence. Recent findings have shown that NATs can exert their regulatory functions by acting as epigenetic regulators of gene expression and chromatin remodeling. Here, we review recent work on the mechanisms of epigenetic modifications by NATs and their emerging role as master regulators of chromatin states. Unlike other long ncRNAs, antisense RNAs usually regulate their counterpart sense mRNA in cis by bridging epigenetic effectors and regulatory complexes at specific genomic loci. Understanding the broad range of effects of NATs will shed light on the complex mechanisms that regulate chromatin remodeling and gene expression in development and disease.

Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells

Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA‐directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin‐remodelling factors. Here, we show that a promoter‐targeted siRNA inhibits transcription of the c‐myc gene. Transcriptional silencing of c‐myc did not involve changes of known epigenetic marks. Instead, the c‐myc promoter‐targeted siRNA interfered with transcription initiation blocking the assembly of the pre‐initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter‐associated RNA initiated upstream and overlapping the transcription start site. Silencing of c‐myc led to growth arrest, reduced clonogenic potential and senescence of c‐myc over‐expressing prostate cancer cells with minimal effect on normal cells. RNA‐directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases.

Modulation of the Activity of Sp Transcription Factors by Mithramycin Analogues as a New Strategy for Treatment of Metastatic Prostate Cancer

Deregulated activity of transcription factors (TFs) of the Sp/KLF family, like Sp1, Sp3 and Sp4, and consequent over-expression of Sp-regulated genes occur frequently in human cancers. This provides the rationale for development of inhibitors of Sp TFs as cancer therapeutics. Mithramycin A (MTM-A) is a natural polyketide that binds GC-rich DNA sequences, inhibits activity of Sp TFs and exhibits potent antitumor activity in experimental systems. However, clinical use of MTM-A is limited by the severe toxicity of the compound. Here, we studied two MTM-A analogues, which had been generated by genetically engineering of the MTM-A biosynthetic pathway, and evaluated their activity in human prostate cancer in cell cultures and mouse models. The compounds, named MTM-SDK and MTM-SK, were highly effective in vitro inhibiting proliferation of prostate cancer cells and transcription of Sp-regulated genes by blocking binding of Sp proteins to the gene promoters When administered to mice, both compounds were well tolerated with maximum tolerated doses of MTM-SDK and MTM-SK, respectively, 4- and 32- fold higher than MTM-A. After systemic administration, both compounds were cleared rapidly from the bloodstream but maintained plasma levels well above the active concentrations required in vitro for inhibition of Sp TF activity and cell proliferation. Consistently, MTM-SDK and MTM-SK inhibited transcription of Sp-regulated genes in prostate tumor xenografts and exhibited potent antitumor activity in subcutaneous and metastatic tumor xenograft models with no or minimal toxicity. Taken together, these data indicate that MTM-SDK and MTM-SK possess significantly improved pharmacological and toxicological properties compared to MTM-A and represent promising drugs for treatment of advanced prostate cancer.

Transcriptomics Profiling of Alzheimer's Disease Reveal Neurovascular Defects, Altered Amyloid-β Homeostasis, and Deregulated Expression of Long Noncoding RNAs

The underlying genetic variations of late-onset Alzheimers disease (LOAD) cases remain largely unknown. A combination of genetic variations with variable penetrance and lifetime epigenetic factors may converge on transcriptomic alterations that drive LOAD pathological process. Transcriptome profiling using deep sequencing technology offers insight into common altered pathways regardless of underpinning genetic or epigenetic factors and thus represents an ideal tool to investigate molecular mechanisms related to the pathophysiology of LOAD. We performed directional RNA sequencing on high quality RNA samples extracted from hippocampi of LOAD and age-matched controls. We further validated our data using qRT-PCR on a larger set of postmortem brain tissues, confirming downregulation of the gene encoding substance P (TAC1) and upregulation of the gene encoding the plasminogen activator inhibitor-1 (SERPINE1). Pathway analysis indicates dysregulation in neural communication, cerebral vasculature, and amyloid-β clearance. Beside protein coding genes, we identified several annotated and non-annotated long noncoding RNAs that are differentially expressed in LOAD brain tissues, three of them are activity-dependent regulated and one is induced by Aβ(1-42) exposure of human neural cells. Our data provide a comprehensive list of transcriptomics alterations in LOAD hippocampi and warrant holistic approach including both coding and non-coding RNAs in functional studies aimed to understand the pathophysiology of LOAD.

Expression of non-protein-coding antisense RNAs in genomic regions related to autism spectrum disorders

BackgroundAutism spectrum disorders (ASD) manifest with neurodevelopmental phenotypes including communicative, social and behavioral impairments that affect as many as 1 in 88 children. The majority of autism cases have no known genetic cause, suggesting complex genetics of the disorder, but a few genes of large effect have been identified.MethodsIn order to identify novel ASD genetic correlates, we investigated non-protein coding RNAs (ncRNAs) which are abundantly transcribed from the human genome, enriched in the brain, and have been implicated in neurodevelopmental disorders. Using an algorithm that we developed, we examined a publicly available transcriptomics database, AceView, to identify the natural antisense transcripts (NATs) that overlap with known autism-related genes. We validated the presence and differential expression of NATs in different brain regions of ASD and control brains using qRT-PCR. Additionally, we investigated the subcellular localization of these transcripts in a neuronal cell line using RNA-sequencing (RNA-seq).ResultsWe found noncoding antisense RNA transcripts at approximately 40% of loci previously implicated in ASD. We confirmed the expression of 10 antisense RNAs in different postmortem human brain tissues. The expression of five antisense transcripts was found to be region-specific, suggesting a role for these ncRNAs in the development and function of specific brain regions. Some antisense RNAs overlapping suspected ASD genes exhibited concordant expression relative to their sense protein-coding genes, while other sense-antisense pairs demonstrate a discordant relationship. Interestingly, the antisense RNA corresponding to the SYNGAP1 locus (SYNGAP1-AS) was found to be differentially expressed in brain regions of patients with ASD compared to control individuals. RNA-seq analysis of subcellular compartments from SH-SY5Y human neuroblastoma cells demonstrated that antisense RNAs to ASD candidate genes are predominantly expressed in the nucleoplasmic or chromatin compartments, implying their involvement in nuclear-associated processes.ConclusionsOur data suggests that NATs are abundantly expressed from ASD-related loci and provide evidence for their roles in target gene regulation, neurodevelopment and autism pathogenesis. This class of RNA should therefore be considered in functional studies aimed at understanding genetic risk factors for ASD.

Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

Long non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimers disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

Upregulation of Haploinsufficient Gene Expression in the Brain by Targeting a Long Non-coding RNA Improves Seizure Phenotype in a Model of Dravet Syndrome

Dravet syndrome is a devastating genetic brain disorder caused by heterozygous loss-of-function mutation in the voltage-gated sodium channel gene SCN1A. There are currently no treatments, but the upregulation of SCN1A healthy allele represents an appealing therapeutic strategy. In this study we identified a novel, evolutionary conserved mechanism controlling the expression of SCN1A that is mediated by an antisense non-coding RNA (SCN1ANAT). Using oligonucleotide-based compounds (AntagoNATs) targeting SCN1ANAT we were able to induce specific upregulation of SCN1A both in vitro and in vivo, in the brain of Dravet knock-in mouse model and a non-human primate. AntagoNAT-mediated upregulation of Scn1a in postnatal Dravet mice led to significant improvements in seizure phenotype and excitability of hippocampal interneurons. These results further elucidate the pathophysiology of Dravet syndrome and outline a possible new approach for the treatment of this and other genetic disorders with similar etiology.

Mechanisms of triplex DNA-mediated inhibition of transcription initiation in cells.

Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF(II)130 and TAF(II)250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells.

The BET-Bromodomain Inhibitor JQ1 Reduces Inflammation and Tau Phosphorylation at Ser396 in the Brain of the 3xTg Model of Alzheimer’s Disease

Abstract: Background Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by well-defined neuropathological brain changes including amyloid plaques, neurofibrillary tangles and the presence of chronic neuroinflammation. Objective: The brain penetrant BET bromodomain inhibitor JQ1 has been shown to regulate inflammation responses in vitro and in vivo, but its therapeutic potential in AD is currently unknown. Method: Three-month-old 3xTg mice were injected once a day with JQ1 (50 mg/kg) or vehicle for 15 weeks. At the end of the treatment learning and memory was assessed using the modified Barnes maze and the Y maze behavioral tests. Tissue from the brain and other organs was collected for molecular evaluation of neuroinflammation tau pathology and amyloid β. 
Results: JQ1 treatment reduced splenomegaly and neuroinflammation in the brain of treated mice where we observed a reduction in the expression of the pro-inflammatory modulators Il-1b, Il-6, Tnfa, Ccl2, Nos2 and Ptgs2. Additionally, JQ1-treated mice showed a reduction of tau phosphorylation at Ser396 in the hippocampus and frontal cortex while total levels of tau remained unaffected. On the other hand, JQ1 did not ameliorate learning and memory deficits in 7-month-old 3xTg mice. Conclusion: Taken together, our data suggest that BET bromodomain inhibitors hold the promise to be used for the treatment of neurological disorders characterized by neuroinflammation.