The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription–polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells.
ABSTRACT Many long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and molecular mechanisms of action remain poorly understood. Paupar , a CNS expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of genome-wide transcriptional regulatory elements. We show here that Paupar transcript directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through formation of a DNA binding ribonucleoprotein complex containing Paupar , KAP1 and the PAX6 transcription factor. Paupar -KAP1 genome-wide co-occupancy reveals a 4-fold enrichment of overlap between Paupar and KAP1 bound sequences. Furthermore, both Paupar and Kap1 loss of function in vivo accelerates lineage progression in the mouse postnatal subventricular zone (SVZ) stem cell niche and disrupts olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans -acting modes of lncRNA-mediated epigenetic regulation, the mechanisms of KAP1 genomic recruitment and identify Paupar and Kap1 as regulators of SVZ neurogenesis.
Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels. Conclusion: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus–host interaction which may be highly relevant to pathogenesis in vivo. Hepatology 2010
Deregulation of the insulin-like growth factor 1 (IGF-1) signaling pathway is a recurrent finding in mouse models and human patients with cerebellar ataxia and thus represents a common pathological cascade in neuronal cell death that may be targeted for therapy. We have previously identified a point mutation in AF4, a transcription cofactor of RNA polymerase II elongation and chromatin remodeling, that causes progressive and highly specific Purkinje cell (PC) death in the ataxic mouse mutant robotic, leading to the accumulation of AF4 in PCs. Here we confirm that the spatiotemporal pattern of PC degeneration in the robotic cerebellum correlates with the specific profile of AF4 upregulation. To identify the underlying molecular pathways, we performed microarray gene expression analysis of PCs obtained by laser capture microdissection (LCM) at the onset of degeneration. Igf-1 was significantly downregulated in robotic PCs compared with wild-type controls before and throughout the degenerative process. Consistently, we observed a decrease in the activation of downstream signaling molecules including type 1 IGF receptor (IGF-1R) and the extracellular signal-regulated kinase (ERK) 1 and ERK2. Chromatin immunoprecipitation confirmed that Igf-1 is a direct and the first validated target of the AF4 transcriptional regulatory complex, and treatment of presymptomatic robotic mice with IGF-1 indeed markedly delayed the progression of PC death. This study demonstrates that small changes in the levels of a single transcriptional cofactor can deleteriously affect normal cerebellum function and opens new avenues of research for the manipulation of the IGF-1 pathway in the treatment of cerebellar ataxia in humans.
Article16 April 2018Open Access Source DataTransparent process The long non-coding RNA Paupar promotes KAP1-dependent chromatin changes and regulates olfactory bulb neurogenesis Ioanna Pavlaki Ioanna Pavlaki orcid.org/0000-0003-2514-0977 Department of Biology and Biochemistry, University of Bath, Bath, UK Search for more papers by this author Farah Alammari Farah Alammari Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Bin Sun Bin Sun Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Neil Clark Neil Clark MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Search for more papers by this author Tamara Sirey Tamara Sirey MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Search for more papers by this author Sheena Lee Sheena Lee Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Dan J Woodcock Dan J Woodcock Warwick Systems Biology Centre, University of Warwick, Coventry, UK Search for more papers by this author Chris P Ponting Chris P Ponting MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Search for more papers by this author Francis G Szele Francis G Szele Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Keith W Vance Corresponding Author Keith W Vance [email protected] orcid.org/0000-0002-3411-8213 Department of Biology and Biochemistry, University of Bath, Bath, UK Search for more papers by this author Ioanna Pavlaki Ioanna Pavlaki orcid.org/0000-0003-2514-0977 Department of Biology and Biochemistry, University of Bath, Bath, UK Search for more papers by this author Farah Alammari Farah Alammari Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Bin Sun Bin Sun Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Neil Clark Neil Clark MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Search for more papers by this author Tamara Sirey Tamara Sirey MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Search for more papers by this author Sheena Lee Sheena Lee Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Dan J Woodcock Dan J Woodcock Warwick Systems Biology Centre, University of Warwick, Coventry, UK Search for more papers by this author Chris P Ponting Chris P Ponting MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK Search for more papers by this author Francis G Szele Francis G Szele Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Search for more papers by this author Keith W Vance Corresponding Author Keith W Vance [email protected] orcid.org/0000-0002-3411-8213 Department of Biology and Biochemistry, University of Bath, Bath, UK Search for more papers by this author Author Information Ioanna Pavlaki1,‡, Farah Alammari2,‡, Bin Sun2, Neil Clark3, Tamara Sirey3, Sheena Lee2, Dan J Woodcock4, Chris P Ponting3, Francis G Szele2 and Keith W Vance *,1 1Department of Biology and Biochemistry, University of Bath, Bath, UK 2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK 3MRC Human Genetics Unit, The Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK 4Warwick Systems Biology Centre, University of Warwick, Coventry, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1225 385106; E-mail: [email protected] The EMBO Journal (2018)37:e98219https://doi.org/10.15252/embj.201798219 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Many long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and mechanisms of action remain poorly understood. Paupar, a CNS-expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of transcriptional regulatory elements in a genome-wide manner. We show here that the Paupar lncRNA directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through the formation of a ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar-KAP1 genome-wide co-occupancy reveals a fourfold enrichment of overlap between Paupar and KAP1 bound sequences, the majority of which also appear to associate with PAX6. Furthermore, both Paupar and Kap1 loss-of-function in vivo disrupt olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans-acting modes of lncRNA-mediated epigenetic regulation and the mechanisms of KAP1 genomic recruitment, and identify Paupar and Kap1 as regulators of neurogenesis in vivo. Synopsis The formation of an RNP complex containing a long non-coding RNA (lncRNA), a chromatin regulator and transcription factor illustrates how a single nuclear lncRNA can regulate transcription of multiple target genes in trans. The CNS-expressed lncRNA Paupar interacts with the TRIM28/TIF1/KAP1 chromatin regulatory protein. Paupar acts in trans to promote KAP1 chromatin occupancy and H3K9me3 deposition at a subset of bound target sites. Paupar regulation in trans requires the formation of a ribonucleoprotein complex containing Paupar, KAP1 and non-KRAB-ZNF transcription factors such as PAX6. Paupar and KAP1 function as regulators of olfactory bulb neurogenesis in vivo. Introduction A subset of nuclear long non-coding RNAs (lncRNAs) have been shown to act as transcription and chromatin regulators using various mechanisms of action. These include local functions close to the sites of lncRNA synthesis (Engreitz et al, 2016) as well as distal modes of action across multiple chromosomes (Chalei et al, 2014; Vance et al, 2014). Moreover, lncRNA regulatory effects may be mediated by the act of lncRNA transcription as well as RNA sequence-dependent interactions with transcription factors and chromatin-modifying proteins (Vance & Ponting, 2014; Rutenberg-Schoenberg et al, 2016). Some lncRNAs have been proposed to act as molecular scaffolds to facilitate the formation of multicomponent ribonucleoprotein regulatory complexes (Tsai et al, 2010; Zhao et al, 2010; Ilik et al, 2013; Maenner et al, 2013; Yang et al, 2014b), whilst others may act to guide chromatin-modifying complexes to specific binding sites genome-wide (Vance & Ponting, 2014). Studies of cis-acting lncRNAs such as Haunt and Hottip have shown that lncRNA transcript accumulation at their sites of expression can effectively recruit regulatory complexes (Yin et al, 2015; Pradeepa et al, 2017). LncRNAs, however, have also been reported to directly bind and regulate genes across multiple chromosomes away from their sites of synthesis (Chu et al, 2011; Chalei et al, 2014; Vance et al, 2014; West et al, 2014; Carlson et al, 2015). By way of contrast, the mechanisms by which such trans-acting lncRNAs mediate transcription and chromatin regulation at distal bound target genes are less clear. LncRNAs show a high propensity to be expressed in various brain regions and cell types relative to other tissues (Mercer et al, 2008, 2010; Ponjavic et al, 2009). The adult neurogenic stem cell-containing mouse subventricular zone (SVZ) generates neurons throughout life, contributes to brain repair and can be stimulated to limit damage, but is also a source of tumours (Bardella et al, 2016; Chang et al, 2016). During SVZ lineage progression, neural stem cells give rise to transit amplifying progenitors which in turn generate neuroblasts that migrate in the rostral migratory stream (RMS) to the olfactory bulbs (OB; Doetsch et al, 1999). The neuroblasts primarily become granule neurons that differentiate by extending long branched dendritic processes towards the glomerular layer (Petreanu & Alvarez-Buylla, 2002). There they integrate into and modulate circuitry connecting peripheral olfactory receptor neurons with the output neurons of the OB (Gheusi et al, 2000; Lledo & Saghatelyan, 2005). It has been estimated that 8,992 lncRNAs are expressed in the SVZ neurogenic system, many of which are differentially expressed during SVZ/OB neurogenesis, suggesting that at least some of these transcripts may play regulatory roles (Ramos et al, 2013). However, only a minority of SVZ expressed lncRNAs have been analysed functionally and the full scope of their molecular mechanisms of action remain poorly understood. Kap1 encodes an essential chromatin regulatory protein that plays a critical role in embryonic development and in adult tissues. Kap1−/− mice die prior to gastrulation while hypomorphic Kap1 mouse mutants display multiple abnormal embryonic phenotypes, including defects in the development of the nervous system (Cammas et al, 2000; Herzog et al, 2011; Shibata et al, 2011). KAP1 interacts with chromatin binding proteins such as HP1 and the SETDB1 histone-lysine N-methyltransferase to control heterochromatin formation and to silence gene expression at euchromatic loci (Iyengar & Farnham, 2011). Despite this fundamental role in epigenetic regulation, the mechanisms of KAP1 genomic targeting are not fully understood. KAP1 does not contain a DNA binding domain but was originally identified through its interaction with members of the KRAB zinc finger (KRAB-ZNF) transcription factor family. Subsequent studies, however, revealed that KRAB–ZNF interactions cannot account for all KAP1 genomic recruitment events. KAP1 preferentially localises to the 3′ end of zinc finger genes as well as to many promoters and intergenic regions in human neuronal precursor cells. A mutant KAP1 protein, however, that is unable to interact with KRAB-ZNFs still binds to promoters, suggesting functionally distinct subdomains (Iyengar et al, 2011). This work points to the presence of alternative, KRAB-ZNF-independent, mechanisms that operate to target KAP1 to a distinct set of genomic binding sites. We reasoned that this may involve specific RNA–protein interactions between KAP1 and chromatin-bound lncRNAs. The CNS-expressed intergenic lncRNA Paupar represents an ideal candidate chromatin-enriched lncRNA with which to further define trans-acting mechanisms of lncRNA-mediated gene and chromatin regulation. Paupar is transcribed upstream from the Pax6 transcription factor gene and acts to control proliferation and differentiation of N2A neuroblastoma cells in vitro (Vance et al, 2014). Paupar regulates Pax6 expression locally, physically associates with PAX6 protein and interacts with distal transcriptional regulatory elements to control gene expression on multiple chromosomes in N2A cells in a dose-dependent manner. Here, we show that Paupar directly interacts with KAP1 in N2A cells and that together they control the expression of a shared set of target genes enriched for regulators of neural proliferation and differentiation. Our findings indicate that Paupar, KAP1 and PAX6 physically associate on chromatin within the regulatory region of shared target genes and that Paupar knockdown reduces both KAP1 chromatin association and histone H3 lysine 9 trimethylation (H3K9me3) at PAX6 co-bound locations. Genome-wide occupancy maps further identified a fourfold enrichment in the overlap between Paupar and KAP1 binding sites on chromatin, the majority of which (73%) are also estimated to be bound by PAX6. Our results also show that both Paupar and KAP1 loss-of-function in vivo disrupt SVZ/OB neurogenesis. We propose that Paupar and Kap1 are novel regulators of neurogenesis in vivo and that Paupar operates as a transcriptional cofactor to promote KAP1-dependent chromatin changes at a subset of bound regulatory elements in trans via association with non-KRAB-ZNF transcription factors such as PAX6. Results Paupar directly binds the KAP1 chromatin regulatory protein in mouse neural cells in culture The lncRNA Paupar binds transcriptional regulatory elements across multiple chromosomes to control the expression of distal target genes in N2A neuroblastoma cells (Vance et al, 2014). Association with transcription factors such as PAX6 assists in targeting Paupar to chromatin sites across the genome. As Paupar depletion does not alter PAX6 chromatin occupancy (Vance et al, 2014), we hypothesised that Paupar may recruit transcriptional cofactors to PAX6 and other neural transcription factors to regulate gene expression. To test this, we sought to identify transcription and chromatin regulatory proteins that bind both Paupar and PAX6 in N2A cells in culture. In vitro-transcribed biotinylated Paupar was therefore immobilised on streptavidin beads and incubated with N2A cell nuclear extract in a pulldown assay. Bound proteins were washed, eluted and identified using mass spectrometry (Fig 1A). This identified a set of 78 new candidate Paupar-associated proteins that do not bind a control RNA of similar size, including 28 proteins with annotated functions in the control of gene expression that might function as transcriptional cofactors (Fig 1B and Dataset EV1). Figure 1. Paupar directly binds the KAP1 chromatin regulatory protein in mouse N2A neuroblastoma cells A. Overview of the pulldown assay. In vitro-transcribed biotinylated Paupar RNA was immobilised on streptavidin beads and incubated with N2A cell nuclear extract. Bound RNA protein complexes were extensively washed and specific Paupar-associated proteins, which do not interact with a control mRNA of a similar size, identified by mass spectrometry. B. Gene Ontology terms were used to annotate Paupar-associated proteins according to biological process. The Bonferroni correction was used to adjust the P-values to correct for multiple testing. C. Endogenous Paupar transcript interacts with transcription and chromatin regulatory proteins in N2A cells. Paupar association with the indicated proteins was measured using native RNA-IP. Whole cell lysates were prepared and the indicated regulatory proteins immuno-precipitated using specific antibodies. Bound RNAs were purified and the levels of Paupar and the U1snRNA control detected in each RIP using qRT–PCR. Paupar transcript directly interacts with KAP1 and RCOR3 in N2A cells. D, E. Nuclear extracts were prepared from UV cross-linked (D) and untreated (E) cells and immuno-precipitated using either anti-KAP1, anti-RCOR3 or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and the U1snRNA control transcript were detected in each UV-RIP using qRT–PCR. F. PAX6 associates with KAP1 in N2A cells. FLAG-PAX6 and KAP1 or RCOR3 expression vectors were transfected into N2A cells. Lysates were prepared 2 days after transfection and FLAG-PAX6 protein immuno-precipitated using anti-FLAG beads. Co-precipitated proteins were detected by Western blotting. Data information: For RNA-IP and UV-RIP assays, results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3. One-tailed t-test, unequal variance *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embj201798219-sup-0007-SDataFig1.pdf] Download figure Download PowerPoint We next performed native RNA-IP experiments in N2A cells to validate potential associations between the endogenous Paupar transcript and five gene expression regulators. These candidates were as follows: RCOR3, a member of the CoREST family of proteins that interact with the REST transcription factor; KAP1, a key epigenetic regulator of gene expression and chromatin structure; PPAN, a previously identified regulator of Pax6 expression in the developing eye; CHE-1, a polymerase II interacting protein that functions to promote cellular proliferation and block apoptosis; and ERH, a transcriptional cofactor that is highly expressed in the eye, brain and spinal cord. The results revealed that the Paupar transcript, but not a non-specific control RNA, was > twofold enriched using antibodies against RCOR3, KAP1, ERH, PPAN or CHE1 compared to an IgG isotype control in a native RNA-IP experiment (Fig 1C). In addition, Paupar did not associate above background with SUZ12, EED and EZH2 Polycomb proteins used as negative controls. This served to further confirm the specificity of the Paupar lncRNA–protein interactions because Polycomb proteins associate with a large number of RNAs (Davidovich et al, 2015) and yet were not identified as Paupar interacting proteins in our pulldown assay. The endogenous Paupar transcript therefore associates with proteins involved in transcription and chromatin regulation in proliferating N2A cells. To characterise Paupar lncRNA–protein interactions further, we used UV-RNA-IP to test whether Paupar interacts directly with any of these five cofactors. These data showed that Paupar, but not an U1snRNA control, is highly enriched using antibodies against KAP1 or RCOR3 compared to an IgG control (Fig 1D). A lower level of Paupar enrichment is found with CHE1, whereas ERH or PPAN does not appear to interact directly with Paupar (Fig EV1). Furthermore, the association of Paupar with either KAP1 or RCOR3 was reduced in the absence of UV treatment (Fig 1E). These results indicate that the endogenous Paupar transcript directly and specifically associates with RCOR3 and KAP1 transcriptional cofactors in neural precursor-like cells in culture. Click here to expand this figure. Figure EV1. Characterisation of Paupar lncRNA–protein interactions using UV-RNA-IPNuclear extracts were prepared from UV cross-linked N2A cells and immuno-precipitated using either the indicated antibodies or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and U1snRNA were detected in each UV-RIP using qRT–PCR. Results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3. Download figure Download PowerPoint As a first step to determine whether KAP1 or RCOR3 can act as PAX6-associated transcriptional cofactors, we performed immunoprecipitation experiments in N2A cells using transfected FLAG-tagged PAX6 and KAP1 or RCOR3 proteins. Immunoprecipitation of FLAG-PAX6 using anti-FLAG beads co-immuno-precipitated transfected KAP1 protein, but not RCOR3 (Fig 1F), suggesting that PAX6 and KAP1 are present within the same multicomponent regulatory complex. Consistent with this, a previous study showed that KAP1 interacts with PAX3 through the amino terminal paired domain, which is structurally similar in PAX6, to mediate PAX3-dependent transcriptional repression (Hsieh et al, 2006). Together, these results indicate that KAP1 may regulate Paupar and PAX6-mediated gene expression programmes. Paupar and KAP1 control expression of a shared set of target genes that are enriched for regulators of neuronal function and cell cycle in N2A cells KAP1 regulates the expression of genes involved in the self-renewal and differentiation of multiple cell types, including neuronal cells (Iyengar & Farnham, 2011), and thus is an excellent candidate interactor for mediating the transcriptional regulatory function of Paupar. To investigate whether Paupar and KAP1 functionally interact to control gene expression, we first tested whether they regulate a common set of target genes. We depleted Kap1 expression in N2A cells using shRNA transfection and achieved ~90% reduction in both protein (Fig 2A) and transcript (Fig 2B) levels. Paupar levels do not change upon KAP1 knockdown, indicating that KAP1-dependent changes in gene expression are not due to regulation of Paupar expression (Fig 2B). Transcriptome profiling using microarrays then identified 1,913 differentially expressed genes whose expression significantly changed [at a 5% false discovery rate (FDR)] greater than 1.4-fold (log2 fold change ≈ 0.5) upon KAP1 depletion (Fig 2C and Dataset EV2). 282 of these genes were previously identified to be regulated by human KAP1 in Ntera2 undifferentiated human neural progenitor cells (Iyengar et al, 2011). Transient reduction in Kap1 expression by ~55% using a second shRNA expression vector (Kap1 shB) also induced expression changes for seven out of eight KAP1 target genes with known functions in neuronal cells that were identified in the microarray (Fig EV2). These data further validate the specificity of the KAP1 regulated gene set. Figure 2. Paupar and KAP1 regulate shared target genes involved in neural cell proliferation and functionN2A cells were transfected with either the shA Kap1 targeting shRNA expression vector or a scrambled control and pTK-Hyg selection plasmid. Three days later, cells were expanded and hygromycin was added to the medium to remove untransfected cells. After 7 days, Western blotting was performed to determine KAP1 protein levels. Lamin B1 was used as a loading control. Kap1 and Paupar transcript levels were analysed by qRT–PCR. Data were normalised using Gapdh, and expression changes are shown relative to a non-targeting scrambled control (set at 1). Mean values ± SEM, N = 3. One-tailed t-test, unequal variance **P < 0.01. KAP1 regulated genes were identified using a GeneChip Mouse Gene 1.0 ST Array (5% FDR, log2 fold change > 0.5). Intersection of Kap1- and Paupar-regulated genes revealed common target genes whose expression is controlled by both these factors. The majority (87%) of Paupar and Kap1 shared target genes are positively regulated by Paupar. Gene Ontology analysis of Paupar and Kap1 common target genes was performed using GOToolBox. Representative significantly enriched categories were selected from a hypergeometric test with a Benjamini–Hochberg-corrected P-value threshold of 0.05. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Validation of the specificity of the KAP1 regulated gene setN2A cells were transfected with an additional Kap1 targeting shRNA expression vector shB-Kap1 or a scrambled control plasmid. Three days later, cells were harvested and expression of the indicated KAP1 targets analysed using RT–qPCR. Samples were normalised using Gapdh, and the results are presented relative to the control. Results are presented as mean values ± SEM, N = 3; *P < 0.05, one-tailed t-test, unequal variance. Download figure Download PowerPoint We previously showed that Paupar knockdown induces changes in the expression of 942 genes in N2A cells (Vance et al, 2014). Examination of the intersection of KAP1 and Paupar transcriptional targets identified 244 genes whose levels are affected by both Paupar and KAP1 knockdown in this cell type (Fig 2D and Dataset EV3). This represents a significant 3.6-fold enrichment over the number expected by random sampling and is not due to co-regulation because Kap1 is not a Paupar target (Vance et al, 2014). A large majority (87%; 212/244) of these common targets are positively regulated by Paupar and for two-thirds of these genes (161/244) their expression changes in the same direction upon Paupar or KAP1 knockdown (Fig 2E). Furthermore, Gene Ontology enrichment analysis of these 244 genes showed that Paupar and KAP1 both regulate a shared set of target genes enriched for regulators of interphase, components of receptor tyrosine kinase signalling pathways as well as genes involved in nervous system development and essential neuronal cell functions such as synaptic transmission (Fig 2F). Genes targeted by both Paupar and KAP1 are thus expected to contribute to the control of neural stem cell self-renewal and neural differentiation. Paupar, KAP1 and PAX6 associate on chromatin within the regulatory region of shared target genes In order to investigate Paupar-mediated mechanisms of distal gene regulation, we next sought to determine whether Paupar, KAP1 and PAX6 can form a ternary complex on chromatin within the regulatory regions of their shared target genes. To do this, we first integrated our analysis of PAX6-regulated gene expression programmes in N2A cells (Vance et al, 2014) and identified 87 of the 244 Paupar and KAP1 common targets, which is 35.8-fold greater than expected by random sampling, whose expression is also controlled by PAX6 (Fig 3A and Dataset EV3). We found that 34 of these genes contain a CHART-Seq mapped Paupar binding site within their GREAT defined putative regulatory regions (Vance et al, 2014; Vance, 2016) and predicted that these represent functional Paupar binding events within close genomic proximity to direct transcriptional target genes (Fig 3A and Dataset EV3). Figure 3. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at PAX6 bound sequences within the regulatory regions of common targets A. Intersection of Paupar, KAP1 and PAX6 regulated genes identified 87 common target genes. 34 of these genes (in brackets) contain a Paupar binding site within their regulatory regions. B. ChIP assays were performed in N2A cells using either an antibody against KAP1 or an isotype-specific control. C. N2A cells were transfected with either a non-targeting control or two independent Paupar targeting shRNA expression vectors. Cells were harvested for ChIP 3 days later, and Paupar depletion was confirmed using qRT–PCR. D. Paupar knockdown reduces KAP1 chromatin occupancy at shared binding sites. ChIP assays were performed 3 days after shRNA transfection using an anti-KAP1 polyclonal antibody. E. Western blotting showed that KAP1 proteins levels do not change upon Paupar knockdown. Actin was used as a control. F. Paupar promotes KAP1–PAX6 association. FLAG-PAX6 and KAP1 expression vectors were co-transfected into N2A cells along with increasing concentrations of Paupar or a size-matched control lncRNA expression vector. Expression of the maximum concentration of either Paupar or control RNA in each IP does not alter KAP1 input protein levels (lower panel). Lysates were prepared 2 days after transfection and FLAG-PAX6 protein immuno-precipitated using anti-FLAG beads. The amount of DNA transfected was made equal in each IP using empty vector and proteins in each complex were detected by Western blotting. G, H. Paupar knockdown reduces H3K9me3 at a subset of bound sequences in trans. ChIP assays were performed using an anti-H3K9me3 polyclonal antibody 3 days after transfection of the indicated shRNA expression vectors. Data information: For ChIP assays, the indicated DNA fragments were amplified using qPCR. % input was calculated as . Results are presented as mean values ± SEM, N = 3. One-tailed t-test, unequal variance *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 3 [embj201798219-sup-0008-SDataFig3.pdf] Download figure Download PowerPoint ChIP-qPCR analysis previously identified four of these Paupar bound locations within the regulatory regions of the Mab21L2, Mst1, E2f2 and Igfbp5 genes that are also bound by PAX6 in N2A cells (Vance et al, 2014). We therefore measured KAP1 chromatin occupancy at these regions as well as at a negative control sequence within the first intron of E2f2 using ChIP and identified a specific enrichment of KAP1 chromatin association at the Mab21L2, Mst1, E2f2 and Igfbp5 genes compared to an IgG isotype control (Fig 3B). KAP1 binding to these regions is only two- to fourfold reduced compared to the Zfp382 3′ UTR-positive control (Fig 3B), which represents an exemplar high-affinity KAP1 binding site (Iyengar et al, 2011). KAP1 and Paupar also co-occupy a binding site within the Ezh2 gene. Ezh2 is regulated by Paupar and KAP1 but not by PAX6 suggesting that transcription factors in addition to PAX6 may also be involved in modulating Paupar-KAP1 function. However, taken together these data indicate that Mab21L2, Mst1, E2f2 and Igfbp5 are co-ordinately regulated by a ribonucleoprotein complex containing Paupar-KAP1–PAX6. Paupar functions as a transcriptional cofactor to promote KAP1 chromatin occupancy and H3K9me3 deposition at PAX6 bound sequences KAP1 is recruited to its target sites within 3′ UTRs of ZNF genes through association with KRAB-ZNF transcription factors (O'Geen et al, 2007; Iyengar et al, 2011). However, Paupar bound sequences are preferentially located at gene promoters and are not enriched for KRAB-ZNF transcription factor binding motifs as determined using de novo motif discovery (Vance et al, 2014). This suggests that Paupar may play a role in recruiting KAP1 to a separate class of binding site in a KRAB-ZNF-independent mann
Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected.
Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.
Abstract Perception is the result of interactions between the sensory periphery, thalamus, and cerebral cortex. Inputs from the retina project to the first‐order dorsal lateral geniculate nucleus (dLGN), which projects to the primary visual cortex (V1). In return, the cortex innervates the thalamus. While layer 6 projections innervate all thalamic nuclei, cortical layer 5 neurons selectively project to the higher order lateral posterior nucleus (LP) and not to dLGN. It has been demonstrated that a subpopulation of layer 5 (Rbp4‐Cre+) projections rewires to dLGN after monocular or binocular enucleation in young postnatal mice. However, the exact cortical regional origin of these projections was not fully determined, and it remained unclear whether these changes persisted into adulthood. In this study, we report gene expression changes observed in the dLGN after monocular enucleation at birth using microarray, qPCR at P6, and in situ hybridization at P8. We report that genes that are normally enriched in dLGN, but not LP during development are preferentially downregulated in dLGN following monocular enucleation. Comparisons with developmental gene expression patters in dLGN suggest more immature and delayed gene expression in enucleated dLGN. Combined tracing and immuno‐histochemical analysis revealed that the induced layer 5 fibers that innervate enucleated dLGN originate from putative primary visual cortex and they retain increased VGluT1+ synapse formation into adulthood. Our results indicate a new form of plasticity when layer 5 driver input takes over the innervation of an originally first‐order thalamic nucleus after early sensory deficit.
Sleep and circadian rhythm disruption has been widely observed in neuropsychiatric disorders including schizophrenia [1Wulff K. Porcheret K. Cussans E. Foster R.G. Sleep and circadian rhythm disturbances: multiple genes and multiple phenotypes.Curr. Opin. Genet. Dev. 2009; 19: 237-246Crossref PubMed Scopus (87) Google Scholar] and often precedes related symptoms [2Ruhrmann S. Schultze-Lutter F. Salokangas R.K. Heinimaa M. Linszen D. Dingemans P. Birchwood M. Patterson P. Juckel G. Heinz A. et al.Prediction of psychosis in adolescents and young adults at high risk: results from the prospective European prediction of psychosis study.Arch. Gen. Psychiatry. 2010; 67: 241-251Crossref PubMed Scopus (558) Google Scholar]. However, mechanistic basis for this association remains unknown. 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Retinal inputs appear normal in mutants, and clock gene rhythms within the suprachiasmatic nucleus (SCN) are normally phased both in vitro and in vivo. However, the 24 hr rhythms of arginine vasopressin within the SCN and plasma corticosterone are both markedly phase advanced in Bdr mice. We suggest that the Bdr circadian phenotype arises from a disruption of synaptic connectivity within the SCN that alters critical output signals. Collectively, our data provide a link between disruption of circadian activity cycles and synaptic dysfunction in a model of neuropsychiatric disease.
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