A novel network pharmacology approach for leukaemia differentiation therapy using Mogrify®.

Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.

Proteomics and Transcriptomics of the Hippocampus and Cortex in SUDEP and High-Risk SUDEP Patients.

OBJECTIVE: To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to control patients with epilepsy. METHODS: For proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected postmortem brain tissue of 12 patients with SUDEP and 14 with non-SUDEP epilepsy. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from patients with mesial temporal lobe epilepsy: 6 low-risk and 8 high-risk SUDEP as determined by a short (<50 seconds) or prolonged (≥50 seconds) postictal generalized EEG suppression (PGES) that may indicate severely depressed brain activity impairing respiration, arousal, and protective reflexes. RESULTS: In autopsy hippocampus and cortex, we observed no proteomic differences between patients with SUDEP and those with non-SUDEP epilepsy, contrasting with our previously reported robust differences between epilepsy and controls without epilepsy. Transcriptomics in hippocampus and cortex from patients with surgical epilepsy segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 long noncoding RNAs, 3 pending) in hippocampus. CONCLUSION: The SUDEP proteome and high-risk SUDEP transcriptome were similar to those in other patients with epilepsy in hippocampus and cortex, consistent with diverse epilepsy syndromes and comorbid conditions associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions, may identify molecular mechanisms of SUDEP.

Cell type-specific circular RNA expression in human glial cells.

The circular transcriptome of human glial cells is an area of neuroscience that has not been thoroughly elucidated. Circular RNAs (circRNAs) have the potential to facilitate the understanding of vast, complex and unknown mechanisms derived from the human transcriptome, including elements of the human brain that are not known and the evolution of the human brain, the complexities of which are not well understood. Moreover, the glial cells have been determined to contribute to human brain evolution. This study presents the first comprehensive analysis of the human brain glia circRNA transcriptome, that is, astrocytes, microglia and oligodendrocytes. After stringent criteria applied to the detection of circRNAs, it was found that the circular transcriptomes of these glia are unique from one another, and hence might be indicative of distinct roles for circRNAs within the brain. This study found 265, 239 and 442 circRNAs comprising the unique circular transcriptome of astrocytes, microglia and oligodendrocytes, respectively. The most abundant circRNAs in these glial cell types are expressed by parent genes co-expressing linear RNAs in low abundance, suggesting spliceosome activity favorable to the back-splicing mechanism instead of canonical splicing activity.

Analysis of the Circular Transcriptome in the Synaptosomes of Aged Mice.

Recently, circular RNAs (circRNAs) have been revealed to be an important non-coding element of the transcriptome. The brain contains the most abundant and widespread expression of circRNA. There are also indications that the circular transcriptome undergoes dynamic changes as a result of brain ageing. Diminished cognitive function with increased age reflects the dysregulation of synaptic function and ineffective neurotransmission through alterations of the synaptic proteome. Here, we present changes in the circular transcriptome in ageing synapses using a mouse model. Specifically, we observed an accumulation of uniquely expressed circular transcripts in the synaptosomes of aged mice compared to young mice. Individual circRNA expression patterns were characterized by an increased abundance in the synaptosomes of young or aged mice, whereas the opposite expression was observed for the parental gene linear transcripts. These changes in expression were validated by RT-qPCR. We provide the first comprehensive survey of the circular transcriptome in mammalian synapses, thereby paving the way for future studies. Additionally, we present 16 genes that express solely circRNAs, without linear RNAs co-expression, exclusively in young and aged synaptosomes, suggesting a synaptic gene network that functions along canonical splicing activity.

Coding and non-coding transcriptome of mesial temporal lobe epilepsy: Critical role of small non-coding RNAs.

Our understanding of mesial temporal lobe epilepsy (MTLE), one of the most common form of drug-resistant epilepsy in humans, is derived mainly from clinical, imaging, and physiological data from humans and animal models. High-throughput gene expression studies of human MTLE have the potential to uncover molecular changes underlying disease pathogenesis along with novel therapeutic targets. Using RNA- and small RNA-sequencing in parrallel, we explored differentially expressed genes in the hippocampus and cortex of MTLE patients who had undergone surgical resection and non-epileptic controls. We identified differentially expressed genes in the hippocampus of MTLE patients and differentially expressed small RNAs across both the cortex and hippocampus. We found significant enrichment for astrocytic and microglial genes among up-regulated genes, and down regulation of neuron specific genes in the hippocampus of MTLE patients. The transcriptome profile of the small RNAs reflected disease state more robustly than mRNAs, even across brain regions which show very little pathology. While mRNAs segregated predominately by brain region for MTLE and controls, small RNAs segregated by disease state. In particular, our data suggest that specific miRNAs (e.g., let-7b-3p and let-7c-3p) may be key regulators of multiple pathways related to MTLE pathology. Further, we report a strong association of other small RNA species with MTLE pathology. As such we have uncovered novel elements that may contribute to the establishment and progression of MTLE pathogenesis and that could be leveraged as therapeutic targets.

Changes in circular RNA expression patterns during human foetal brain development.

Circular RNAs (circRNAs) are a recently identified class of long non-coding RNAs and their expression is regulated in a tissue- and developmental stage-specific manner. Recent studies indicate the potential regulatory role that circRNAs may have, particularly in the brain, where they are most abundant. This study aims to elucidate changes in circRNA patterns during human embryonic brain development. We detected a number of differentially expressed genes that showed distinct expression profiles for circular and linear transcripts despite having originated from the same genes, implicating a dichotomy in the regulation of these two RNA species. Altogether our study showed that circular and linear RNAs have independent expression patterns, and that circular transcriptomes from different developing stages have distinct characteristics in terms of transcript abundance and isoform diversity.

Early transcriptome changes in response to chemical long-term potentiation induced via activation of synaptic NMDA receptors in mouse hippocampal neurons.

Long term potentiation (LTP) is a form of synaptic plasticity. In the present study LTP was induced via activation of synaptic NMDA receptors in primary hippocampal neuron cultures from neonate mice and RNA was isolated for RNA sequencing at 20 min following LTP induction. RNA sequencing and differential expression testing was performed to determine the identity and abundance of protein-coding and non-coding RNAs in control and LTP induced neuron cultures. We show that expression levels of a small group of transcripts encoding proteins involved in negative regulation of gene expression (Adcyap1, Id3), protein translation (Rpl22L1), extracellular structure organization (Bgn), intracellular signalling (Ppm1H, Ntsr2, Cldn10) and protein citrullination (PAD2) are downregulated in the stimulated neurons. Our results suggest that the early stages of LTP are accompanied by the remodelling of the biosynthetic machinery, interactions with the extracellular matrix and intracellular signalling pathways at the transcriptional level.

Tropomyosin isoforms have specific effects on the transcriptome of undifferentiated and differentiated B35 neuroblastoma cells.

Tropomyosins, a family of actin-associated proteins, bestow actin filaments with distinct biochemical and physical properties which are important for determining cell shape and regulating many cellular processes in eukaryotic cells. Here, we used RNA-seq to investigate the effect of four tropomyosin isoforms on gene expression in undifferentiated and differentiated rat B35 neuroblastoma cells. In undifferentiated cells, overexpression of tropomyosin isoforms Tpm1.12, Tpm2.1, Tpm3.1, and Tpm4.2 differentially regulates a vast number of genes, clustering into several gene ontology terms. In differentiated cells, tropomyosin overexpression exerts a much weaker influence on overall gene expression. Our findings are particularly compelling because they demonstrate that tropomyosin-dependent changes are attenuated once the cells are induced to follow a defined path of differentiation. Database: Sequence data for public availability are deposited in the European Nucleotide Archive under the accession number PRJEB24136.

Analysis of the circular RNA transcriptome in endometrial cancer.

Circular RNAs (circRNAs) are a naturally occurring family of non-coding RNA that may regulate gene expression in mammals. circRNAs are more stable than messenger RNAs due to their resistance to RNA exonuclease. A growing body of evidence has shown that the expression of circRNAs is regulated during development in a tissue-specific manner. CircRNAs have been implicated in a number of cancers; however, their role in endometrial cancer (EC) is completely unknown. Here, we report the circular transcriptome specific for EC as determined by RNA sequencing. We found that the overall abundance of circRNAs is lower in EC than in normal endometrium. Further, there are numerous 'hotspot' genes from which circRNAs are transcribed that may account for alterations in circRNA expression between the normal and malignant endometrium. Most importantly, we have also identified circRNAs that are differentially expressed between malignant and normal endometrial tissue. The functional significance of these circRNAs in cancer remains to be determined, but they may serve as potential biomarkers for the diagnosis of EC or monitoring of EC progression.

Multiple System Atrophy: Many Lessons from the Transcriptome.

Multiple system atrophy (MSA) is a complex, multifactorial, debilitating neurodegenerative disease that is often misdiagnosed and misunderstood. MSA has two subclasses, MSA-P and MSA-C, defined by the dominance of parkinsonism or cerebellar dysfunction in the earlier stages of disease, coupled with dysautonomia. This distinction between subclasses becomes largely redundant as the disease progresses. Aggregation of α-synuclein is a clinical marker used to confirm MSA diagnoses, which can only be performed postmortem. Transcriptome profiling provides in-depth information about the diseased state and can contribute to further understanding of MSA, enabling easier and more rapid diagnosis as well as contributing to improving the quality of life of people with MSA. Currently, there is no method of diagnosing MSA with certainty, and there is no cure for this disease. This review provides an update on current advances in investigations of molecular pathology of MSA with particular focus on perturbation of individual gene expression and MSA transcriptome.

Region-specific expression of circular RNAs in the mouse brain.

Circular RNAs (circRNAs) are abundant in mammalian brain and their expression is regulated in a tissue- and developmental stage-specific manner. Mammalian brain is the most transcriptionally complex organ. While many studies have extensively studied linear transcriptome and its biological functions in the brain, the circular transcriptome remains largely unexplored. This study focused on investigation of circRNA expression patterns in the mammalian brain regions critical for cognitive and memory functions and performed comparative analysis with the linear transcriptome. Altogether our study showed that circular and linear RNAs have independent expression patterns despite being derived from the same genomic locus, and that circular transcriptomes from different brain region have distinct characteristics in terms of transcript abundance and composition.

Transcriptome landscape of long intergenic non-coding RNAs in endometrial cancer.

Endometrial cancer is the most common gynecological malignancy in the developed world. It is the fifth most common cancer and accounts for 4.8% of all cancers in women. Long intergenic non-coding RNAs (lincRNAs), a subclass of long non-coding RNAs, are pervasively transcribed throughout the human genome. OBJECTIVE: LincRNA expression patterns in endometrial cancer compared to normal healthy tissue are poorly characterised. In this study, the lincRNA transcriptome of endometrial cancers and adjacent normal endometrium from the same patients was sequenced and compared with transcriptomes of other gynaecologic malignancies including ovarian and cervical cancers. METHODS: RNA was isolated from malignant and adjacent non-affected endometrial tissue from 6 patients with low grade and stage Type I endometrial cancer. Subsequently, Illumina paired-end RNA sequencing was performed, followed by bioinformatics analysis, to determine differential transcriptome expression patterns. RESULTS: LINC00958 was upregulated in all three cancers, and four lincRNAs including LINC01480, LINC00645, LINC00891 and LINC00702 demonstrated exquisite specificity for malignant endometrium compared to normal endometrium while also distinguishing endometrial cancer from ovarian and cervical cancers. Furthermore, LINC01480 has features required to express a micropeptide. CONCLUSIONS: The lincRNAs, characterised in this study, represent high priority genes to be tested for functional significance in the pathogenesis and/or progression of endometrial cancer. Furthermore, lincRNAs have potential to be released into the bloodstream and therefore the four lincRNAs identified here may represent biomarkers for early detection of endometrial cancer without biopsy.

RNA sequencing reveals pronounced changes in the noncoding transcriptome of aging synaptosomes.

Normal aging is associated with impairments in cognitive functions. These alterations are caused by diminutive changes in the biology of synapses, and ineffective neurotransmission, rather than loss of neurons. Hitherto, only a few studies, exploring molecular mechanisms of healthy brain aging in higher vertebrates, utilized synaptosomal fractions to survey local changes in aging-related transcriptome dynamics. Here we present, for the first time, a comparative analysis of the synaptosomes transcriptome in the aging mouse brain using RNA sequencing. Our results show changes in the expression of genes contributing to biological pathways related to neurite guidance, synaptosomal physiology, and RNA splicing. More intriguingly, we also discovered alterations in the expression of thousands of novel, unannotated lincRNAs during aging. Further, detailed characterization of the cleavage and polyadenylation factor I subunit 1 (Clp1) mRNA and protein expression indicates its increased expression in neuronal processes of hippocampal stratum radiatum in aging mice. Together, our study uncovers a new layer of transcriptional regulation which is targeted by aging within the local environment of interconnecting neuronal cells.

The emerging role of long non-coding RNAs in endometrial cancer.

The human genome is pervasively transcribed and approximately 98% of the genome is non-coding. Long non-coding RNAs (lncRNAs) are a heterogeneous group of RNA transcripts that are >200 nucleotides in length with minimal to no protein-coding potential. Similar to proteins, lncRNAs have important biological functions in both normal cells and disease states including many types of cancer. This review summarizes recent advances in our understanding of lncRNAs in cancer biology and highlights the potential for lncRNA as diagnostic biomarkers and therapeutics. Herein we focus on the poorly understood role of lncRNAs in endometrial cancer, the most common gynecologic malignancy in the developed world.

Characterization of circular RNAs landscape in multiple system atrophy brain.

Circular RNAs (circRNAs) have been recently identified as a naturally occurring family of widespread and diverse endogenous non-coding RNAs that may regulate gene expression in mammals. They are unusually stable RNA molecules with cell type- or developmental stage-specific expression patterns. However, the role of circRNAs in pathology of complex disease is entirely unknown. Here, we report the specific circular transcriptome in the multiple system atrophy (MSA) brain as determined by RNA sequencing. Five circRNAs, namely IQCK, MAP4K3, EFCAB11, DTNA, and MCTP1, were identified and validated as specifically over-expressed in MSA frontal cortex. The expression levels of linear transcripts were not significantly altered and thus did not follow the pattern of their circular counterparts. Further analysis of expression of five MSA-specific circRNAs revealed their over-expression in the white matter of the MSA cortical tissue. Together, this is the first report describing perturbation of circular transcriptome in α-synucleinopathies.

LINC00507 Is Specifically Expressed in the Primate Cortex and Has Age-Dependent Expression Patterns.

Over the past decade, there has been an increase in the appreciation of the role of non-coding RNA in the development of organism phenotype. It is possible to divide the non-coding elements of the transcriptome into three categories: short non-coding RNAs, circular RNAs and long non-coding RNAs. Long non-coding RNAs are those transcripts that are greater than 200 nts in length and lack any significant open reading frames that produce proteins greater then 100 amino acids. Long intervening non-coding RNAs (lincRNAs) are a subclass of long non-coding RNAs. In contrast to protein coding RNAs, lincRNAs are expressed in a more tissue- and species-specific manner. In particular, many lincRNAs are only conserved amongst higher primates. This coupled with the propensity of many lincRNAs to be expressed in the brain, suggests that they are in fact one of the major drivers of organism complexity. We analysed 39 lincRNAs that are expressed in the frontal cortex and identified LINC00507 as being expressed in a cortex-specific manner in non-human primates and humans. The expression patterns of LINC00507 appear to be age-dependent, suggesting it may be involved in brain development of higher primates. Moreover, the analysis of LINC00507 potential to bind ribosomes revealed that this previously identified non-coding transcript may harbour a micropeptide.

RNA-Sequencing to Elucidate Early Patterns of Dysregulation Underlying the Onset of Alzheimer's Disease.

With its ability to perform rapid transcriptome profiling and profound transcriptomic analysis powered by high-throughput sequencing at a high resolution with deep coverage, the advent of RNA sequencing technology, RNA-Seq, outperforms other methods in the field, such as microarrays, and has changed our way of performing transcriptomic investigation. Protocols for preparing libraries for RNA-Seq using the Illumina and Roche 454 sequencing platforms are included in this chapter. Common steps for library preparation in both platforms include RNA fragmentation, cDNA synthesis, adaptor ligation, and PCR amplification of cDNA strands. Illumina adopts solid-phase bridge PCR amplification, while 454 uses water-in-oil emulsion-based PCR amplification. Despite differences in the PCR amplification step, both platforms employ the same sequencing-by-synthesis technology for the sequencing process. Application of the RNA-Seq technique in the context of dysregulation of the transcriptome in Alzheimer's disease is also discussed.

The Antisense Transcriptome and the Human Brain.

The transcriptome of a cell is made up of a varied array of RNA species, including protein-coding RNAs, long non-coding RNAs, short non-coding RNAs, and circular RNAs. The cellular transcriptome is dynamic and can change depending on environmental factors, disease state and cellular context. The human brain has perhaps the most diverse transcriptome profile that is enriched for many species of RNA, including antisense transcripts. Antisense transcripts are produced when both the plus and minus strand of the DNA helix are transcribed at a particular locus. This results in an RNA transcript that has a partial or complete overlap with an intronic or exonic region of the sense transcript. While antisense transcription is known to occur at some level in most organisms, this review focuses specifically on antisense transcription in the brain and how regulation of genes by antisense transcripts can contribute to functional aspects of the healthy and diseased brain. First, we discuss different techniques that can be used in the identification and quantification of antisense transcripts. This is followed by examples of antisense transcription and modes of regulatory function that have been identified in the brain.

High expression of long intervening non-coding RNA OLMALINC in the human cortical white matter is associated with regulation of oligodendrocyte maturation.

BACKGROUND: Long intervening non-coding RNAs (lincRNAs) are a recently discovered subclass of non-coding RNAs. LincRNAs are expressed across the mammalian genome and contribute to the pervasive transcription phenomenon. They display a tissue-specific and species-specific mode of expression and are present abundantly in the brain. RESULTS: Here, we report the expression patterns of oligodendrocyte maturation-associated long intervening non-coding RNA (OLMALINC), which is highly expressed in the white matter (WM) of the human frontal cortex compared to the grey matter (GM) and peripheral tissues. Moreover, we identified a novel isoform of OLMALINC that was also up-regulated in the WM. RNA-interference (RNAi) knockdown of OLMALINC in oligodendrocytes, which are the major cell type in the WM, caused significant changes in the expression of genes regulating cytostructure, cell activation and membrane signaling. Gene ontology enrichment analysis revealed that over 10% of the top 25 up- and down-regulated genes were involved in oligodendrocyte maturation. RNAi experiments in neuronal cells resulted in the perturbation of genes controlling cell proliferation. Furthermore, we identified a novel cis-natural antisense non-coding RNA, which we named OLMALINC-AS, which maps to the first exon of the dominant isoform of OLMALINC. CONCLUSIONS: Our study has demonstrated for the first time that a primate-specific lincRNA regulates the expression of genes critical to human oligodendrocyte maturation, which in turn might be regulated by an antisense counterpart.

Unique transcriptome patterns of the white and grey matter corroborate structural and functional heterogeneity in the human frontal lobe.

The human frontal lobe has undergone accelerated evolution, leading to the development of unique human features such as language and self-reflection. Cortical grey matter and underlying white matter reflect distinct cellular compositions in the frontal lobe. Surprisingly little is known about the transcriptomal landscape of these distinct regions. Here, for the first time, we report a detailed transcriptomal profile of the frontal grey (GM) and white matter (WM) with resolution to alternatively spliced isoforms obtained using the RNA-Seq approach. We observed more vigorous transcriptome activity in GM compared to WM, presumably because of the presence of cellular bodies of neurons in the GM and RNA associated with the nucleus and perinuclear space. Among the top differentially expressed genes, we also identified a number of long intergenic non-coding RNAs (lincRNAs), specifically expressed in white matter, such as LINC00162. Furthermore, along with confirmation of expression of known markers for neurons and oligodendrocytes, we identified a number of genes and splicing isoforms that are exclusively expressed in GM or WM with examples of GABRB2 and PAK2 transcripts, respectively. Pathway analysis identified distinct physiological and biochemical processes specific to grey and white matter samples with a prevalence of synaptic processes in GM and myelination regulation and axonogenesis in the WM. Our study also revealed that expression of many genes, for example, the GPR123, is characterized by isoform switching, depending in which structure the gene is expressed. Our report clearly shows that GM and WM have perhaps surprisingly divergent transcriptome profiles, reflecting distinct roles in brain physiology. Further, this study provides the first reference data set for a normal human frontal lobe, which will be useful in comparative transcriptome studies of cerebral disorders, in particular, neurodegenerative diseases.