No abstract

Bromodomain and extraterminal (BET) domain proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule inhibitors of BET bromodomain proteins reduce expression of several oncogenes required for Glioblastoma Multiforme (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression, but mechanistic insight into their expression and regulation by BET bromodomain inhibitors remains elusive. In this study, we used Helicos single molecule sequencing to comprehensively profile lncRNAs differentially expressed in GBM, and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET bromdomain inhibitor I-BET151 reduced levels of the tumorpromoting lncRNA HOX transcript antisense RNA (HOTAIR) and restored the expression of several other GBM down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET151 treatment abrogates the antiproliferative activity of the BET bromodomain inhibitor. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Bromodomain Containing 4 (BRD4) to the HOTAIR promoter, suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that modulation of lncRNA networks may, in part, mediate the antiproliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.glioblastoma | long noncoding RNAs | epigenetics | BRD4 | I-BET151

We investigated the postnatal effects of embryonic knockdown and overexpression of the candidate dyslexia gene homolog Kiaa0319. We used in utero electroporation to transfect cells in E15/16 rat neocortical ventricular zone with either 1) small hairpin RNA (shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression construct, 3) Kiaa0319 shRNA along with KIAA0319 expression construct ("rescue"), or 4) a scrambled version of Kiaa0319 shRNA. Knockdown, but not overexpression, of Kiaa0319 resulted in periventricular heterotopias that contained large numbers of both transfected and non-transfected neurons. This suggested that Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as well as non-cell autonomous mechanisms. Of the Kiaa0319 shRNA-transfected neurons that migrated into the cortical plate, most migrated to their appropriate lamina. In contrast, neurons transfected with the KIAA0319 expression vector attained laminar positions subjacent to their expected positions. Neurons transfected with Kiaa0319 shRNA exhibited apical, but not basal, dendrite hypertrophy, which was rescued by overexpression of KIAA0319. The results provide additional supportive evidence linking candidate dyslexia susceptibility genes to migrational disturbances during brain development, and extends the role of Kiaa0319 to include growth and differentiation of dendrites.

Embryonic knockdown of candidate dyslexia susceptibility gene (CDSG) homologs in cerebral cortical progenitor cells in the rat results in acute disturbances of neocortical migration. In the current report we investigated the effects of embryonic knockdown and overexpression of the homolog of DCDC2, one of the CDSGs, on the postnatal organization of the cerebral cortex. Using a within-litter design, we transfected cells in rat embryo neocortical ventricular zone around embryonic day (E) 15 with either 1) small hairpin RNA (shRNA) vectors targeting Dcdc2, 2) a DCDC2 overexpression construct, 3) Dcdc2 shRNA along with DCDC2 overexpression construct, 4) an overexpression construct composed of the C terminal domain of DCDC2, or 5) an overexpression construct composed of the DCX terminal domain of DCDC2. RNAi of Dcdc2 resulted in pockets of heterotopic neurons in the periventricular region. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Dcdc2 shRNA-transfected neurons migrated in a bimodal pattern, with approximately 7% of the neurons migrating a short distance from the ventricular zone, and another 30% migrating past their expected lamina. Rats transfected with Dcdc2 shRNA along with the DCDC2 overexpression construct rescued the periventricular heterotopia phenotype, but did not affect the percentage of transfected neurons that migrate past their expected laminar location. There were no malformations associated with any of the overexpression constructs, nor was there a significant laminar disruption of migration. These results support the claim that knockdown of Dcdc2 expression results in neuronal migration disorders similar to those seen in the brains of dyslexics.

The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5′-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called “Deep-RACE”, which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3′ region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-013-1356-6) contains supplementary material, which is available to authorized users.

To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles’ (MNPs) effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo.

Triplet repeat expansions in the Fragile X mental retardation 1 (FMR1) gene cause either intellectual disability and autism, or adult-onset neurodegeneration, with poorly understood variability in presentation. Previous studies have identified several long noncoding RNAs (lncRNAs) at the FMR1 locus, including FMR4. Similarly to FMR1, FMR4 is silenced by large-repeat expansions that result in enrichment of DNA and histone methylation within the shared promoter and repeat sequence, suggesting a possible role for this noncoding RNA in the pathophysiology of Fragile X. We therefore assessed the functional role of FMR4 to gain further insight into the molecular processes in Fragile X-associated disorders. Previous work showed that FMR4 does not exhibit cis-regulation of FMR1. Here, we found that FMR4 is a chromatin-associated transcript and, using genome-wide chromatin immunoprecipitation experiments, showed that FMR4 alters the chromatin state and the expression of several hundred genes in trans. Among the genes regulated by FMR4, we found enrichment for those involved in neural development and cellular proliferation. S-phase marker assays further demonstrated that FMR4 may promote cellular proliferation, rather than differentiation, of human neural precursor cells (hNPCs). By establishing this novel function for FMR4 in hNPCs, we lend support to existing evidence of the epigenetic involvement of lncRNA in nervous system development, and increase our understanding of the complex pathogenesis underlying neurological disorders associated with FMR1 repeat expansions.

CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. “Full”-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.