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Noel J Buckley

Publications and source records attributed to Noel J Buckley.

15 recordsLinked to original sources
PubMed2006

RE1 Silencing transcription factor maintains a repressive chromatin environment in embryonic hippocampal neural stem cells.

The control of gene expression in neural stem cells is key to understanding their developmental and therapeutic potential, yet we know little of the transcriptional mechanisms that underlie their differentiation. Recent evidence has implicated the RE1 silencing transcription factor (REST) in neuronal differentiation. However, the means by which REST regulates transcription in neural stem cells remain unclear. Here, we show that REST recruits distinct corepressor platforms in neural stem cells. REST is able to both silence and repress neuronal genes in embryonic hippocampal neural stem cells by creating a chromatin environment that contains both repressive local epigenetic signature (characterized by low levels of histones H4 and H3K9 acetylation and elevated dimethylation of H3K9) and H3K4 methylation, which are characteristic of gene activation. Furthermore, inhibition of REST function leads to activation of several neuron-specific genes but does not lead to overt formation of mature neurons, supporting the notion that REST regulates part, but not all, of the neuronal differentiation program.

Animals↗
PubMed2006

BRG1 chromatin remodeling activity is required for efficient chromatin binding by repressor element 1-silencing transcription factor (REST) and facilitates REST-mediated repression.

Chromatin remodeling enzymes such as SWI/SNF use the hydrolysis of ATP to power the movement of nucleosomes with respect to DNA. BRG1, one of the ATPases of the SWI/SNF complex, can be recruited by both activators and repressors, although the precise role of BRG1 in mechanisms of repression has thus far remained unclear. One transcription factor that recruits BRG1 as a corepressor is the repressor element 1-silencing transcription factor (REST). Here we address for the first time the mechanism of BRG1 activity in gene repression. We found that BRG1 enhanced REST-mediated repression at some REST target genes by increasing the interaction of REST with the local chromatin at its binding sites. Furthermore, REST-chromatin interactions, mediated by BRG1, were enhanced following an increase in histone acetylation in a manner dependent on the BRG1 bromodomain. Our data suggest that BRG1 facilitates REST repression by increasing the interaction between REST and chromatin. Such a mechanism may be applicable to other transcriptional repressors that utilize BRG1.

Acetylation↗
PubMed2006

Identification of the REST regulon reveals extensive transposable element-mediated binding site duplication.

The genome-wide mapping of gene-regulatory motifs remains a major goal that will facilitate the modelling of gene-regulatory networks and their evolution. The repressor element 1 is a long, conserved transcription factor-binding site which recruits the transcriptional repressor REST to numerous neuron-specific target genes. REST plays important roles in multiple biological processes and disease states. To map RE1 sites and target genes, we created a position specific scoring matrix representing the RE1 and used it to search the human and mouse genomes. We identified 1301 and 997 RE1s inhuman and mouse genomes, respectively, of which >40% are novel. By employing an ontological analysis we show that REST target genes are significantly enriched in a number of functional classes. Taking the novel REST target gene CACNA1A as an experimental model, we show that it can be regulated by multiple RE1s of different binding affinities, which are only partially conserved between human and mouse. A novel BLAST methodology indicated that many RE1s belong to closely related families. Most of these sequences are associated with transposable elements, leading us to propose that transposon-mediated duplication and insertion of RE1s has led to the acquisition of novel target genes by REST during evolution.

Animals↗
PubMed2006

Stimulation of Galphaq-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK.

Spectrin is a cytoskeletal protein that plays a role in formation of the specialized plasma membrane domains. However, little is known of the molecular mechanism that regulates responses of spectrin to extracellular stimuli, such as activation of G-protein-coupled receptor (GPCR). We have found that alphaII spectrin is a component of the Galpha(q/11)-associated protein complex in CHO cells stably expressing the M1 muscarinic receptor, and investigated the effect of activation of GPCR on the cellular localization of yellow-fluorescent-protein-tagged alphaII spectrin. Stimulation of Galpha(q/11)-coupled M1 muscarinic receptor triggered reversible redistribution of alphaII spectrin following a rise in intracellular Ca2+ concentration. This redistribution, accompanied by non-apoptotic membrane blebbing, required an intact actin cytoskeleton and was dependent on activation of phospholipase C, protein kinase C, and Rho-associated kinase ROCK. Muscarinic-agonist-induced spectrin remodeling appeared particularly active at localized domains, which is clear contrast to that caused by constitutive activation of ROCK and to global rearrangement of the spectrin lattice caused by changes in osmotic pressure. These results suggest a role for spectrin in providing a dynamic and reversible signaling platform to the specific domains of the plasma membrane in response to stimulation of GPCR.

Animals↗
PubMed2006

The transcriptional repressor REST is a critical regulator of the neurosecretory phenotype.

Release of distinct cellular cargoes in response to specific stimuli is a process fundamental to all higher eukaryotes and controlled by the regulated secretory pathway (RSP). However, the mechanism by which genes involved in the RSP are selectively expressed, leading to the establishment and appropriate functioning of regulated secretion remaining largely unknown. Using the rat pheochromocytoma cell line PC12, we provide evidence that, by controlling expression of many genes involved in the RSP, the transcriptional repressor REST can regulate this pathway and hence the neurosecretory phenotype. Introduction of REST transgenes into PC12 cells leads to the repression of many genes, the products of which are involved in regulated secretion. Moreover, chromatin immunoprecipitation assays show that many of the repressed genes recruit the recombinant REST protein to RE1 sites within their promoters and abrogation of REST function leads to reactivation of these transcripts. In addition to the observed transcriptional effects, PC12 cells expressing REST have fewer secretory granules and a reduction in the ability to store and release noradrenaline. Furthermore, an important trigger for synaptic release, influx of calcium through voltage-operated calcium channels, is compromised. This is the first demonstration of a transcription factor that directly controls expression of many major components of the RSP and provides further insight into the function of REST.

Animals↗
PubMed2005

Downregulated REST transcription factor is a switch enabling critical potassium channel expression and cell proliferation.

Induction of K(Ca)3.1 (IKCa) potassium channel plays an important role in vascular smooth muscle cell proliferation. Here, we report that the gene encoding K(Ca)3.1 (KCNN4) contains a functional repressor element 1-silencing transcription factor (REST or NRSF) binding site and is repressed by REST. Although not previously associated with vascular smooth muscle cells, REST is present and recruited to the KCNN4 gene in situ. Significantly, expression of REST declines when there is cellular proliferation, showing an inverse relationship with functional K(Ca)3.1. Downregulated REST and upregulated K(Ca)3.1 are also evident in smooth muscle cells of human neointimal hyperplasia grown in organ culture. Furthermore, inhibition of K(Ca)3.1 suppresses neointimal formation, and exogenous REST reduces the functional impact of K(Ca)3.1. Here, we show REST plays a previously unrecognized role as a switch regulating potassium channel expression and consequently the phenotype of vascular smooth muscle cells and human vascular disease.

Animals↗
PubMed2005

Distinct profiles of REST interactions with its target genes at different stages of neuronal development.

Differentiation of pluripotent embryonic stem (ES) cells through multipotent neural stem (NS) cells into differentiated neurons is accompanied by wholesale changes in transcriptional programs. One factor that is present at all three stages and a key to neuronal differentiation is the RE1-silencing transcription factor (REST/NRSF). Here, we have used a novel chromatin immunoprecipitation-based cloning strategy (SACHI) to identify 89 REST target genes in ES cells, embryonic hippocampal NS cells and mature hippocampus. The gene products are involved in all aspects of neuronal function, especially neuronal differentiation, axonal growth, vesicular transport and release, and ionic conductance. Most target genes are silent or expressed at low levels in ES and NS cells, but are expressed at much higher levels in hippocampus. These data indicate that the REST regulon is specific to each developmental stage and support the notion that REST plays distinct roles in regulating gene expression in pluripotent ES cells, multipotent NS cells, and mature neurons.

Animals↗
PubMed2004

Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

Animals↗
PubMed2004

Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes.

The completion of whole genome sequencing projects has provided the genetic instructions of life. However, whereas the identification of gene coding regions has progressed, the mapping of transcriptional regulatory motifs has moved more slowly. To understand how distinct expression profiles can be established and maintained, a greater understanding of these sequences and their trans-acting factors is required. Herein we have used a combined in silico and biochemical approach to identify binding sites [repressor element 1/neuron-restrictive silencer element (RE1/NRSE)] and potential target genes of RE1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) within the human, mouse, and Fugu rubripes genomes. We have used this genome-wide analysis to identify 1,892 human, 1,894 mouse, and 554 Fugu RE1/NRSEs and present their location and gene linkages in a searchable database. Furthermore, we identified an in vivo hierarchy in which distinct subsets of RE1/NRSEs interact with endogenous levels of REST/NRSF, whereas others function as bona fide transcriptional control elements only in the presence of elevated levels of REST/NRSF. These data show that individual RE1/NRSE sites interact differentially with REST/NRSF within a particular cell type. This combined bioinformatic and biochemical approach serves to illustrate the selective manner in which a transcription factor interacts with its potential binding sites and regulates target genes. In addition, this approach provides a unique whole-genome map for a given transcription factor-binding site implicated in establishing specific patterns of neuronal gene expression.

Animals↗
PubMed2004

Transcription of the M1 muscarinic receptor gene in neurons and neuronal progenitors of the embryonic rat forebrain.

Development of the nervous system is accompanied by expansion and differentiation of the neuronal progenitors within the embryonic neuroepithelium. Although the role of growth factors in this process is well documented, there is increasing evidence for a role of neurotransmitters. Acetylcholine is known to exert many actions on developing neural cells, but its potential role in neurogenesis is unclear. Here, we show that the M1 muscarinic acetylcholine receptor is expressed in the neuroepithelium of the rat forebrain, where it is found on both nestin+ progenitor cells and TuJ1+ newly differentiated neurons. Furthermore, transcription is governed, at least in part, by regulatory cis elements that are also responsible for driving transcription in neuroblastoma cells. This represents the first demonstration of M1 receptors on neuronal progenitor cells and supports the notion that M1 muscarinic receptors may play a role in development of the nervous system prior to the onset of synaptogenesis and their subsequent role in neurotransmission.

Animals↗
PubMed2004

Multiple promoters drive tissue-specific expression of the human M muscarinic acetylcholine receptor gene.

Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5' untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR-32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA-box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA-4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.

Amino Acid Sequence↗
PubMed2003

Interaction of the repressor element 1-silencing transcription factor (REST) with target genes.

The repressor element 1-silencing transcription factor (REST) has been proposed to restrict expression of repressor element 1 (RE1) bearing genes to differentiated neurons by silencing their expression in non-neural tissue. Here, we have examined the interaction of REST with the M(4) muscarinic acetylcholine receptor gene. We show that REST binds to the RE1 of the M(4) gene in those cell lines and brain regions where the M(4) gene is expressed but not in those where the M(4) is not expressed. Furthermore, in cells that express M(4), the presence of REST represses but is insufficient to silence transcription of M(4). In non-neural cells REST is absent from the RE1 of the silent M(4) gene and perturbation of REST function fails to induce M(4) expression. We propose that REST acts to regulate expression levels of some RE1-bearing genes in neural cells, thereby playing an important role in defining neuronal activity.

Base Sequence↗
PubMed2003

Distinct RE-1 silencing transcription factor-containing complexes interact with different target genes.

Establishment of neuronal identity requires co-ordinated expression of specific batteries of genes. These programs of gene expression are executed by activation of neuron-specific genes in neuronal cells and their repression in non-neuronal cells. Such co-ordinate regulation requires that individual activators and repressors regulate transcription from specific subsets of their potential target genes, yet we know little of the mechanisms that underlie this selective process. The RE-1 silencing transcription factor (REST) is a repressor that is proposed to silence transcription of numerous neuron-specific genes in non-neuronal cells via recruitment of two independent histone deacetylase (HDAC)-containing co-repressor complexes. However, in vivo, REST appears to be an obligate silencer for only a minority of RE-1-bearing genes. Here we examine the interaction of REST, Co-REST, Sin3A, HDAC1, and HDAC2 with two archetypical endogenous target genes, the M4 muscarinic receptor and the sodium type II channel (NaV1.2) genes. We find that these genes are present in distinct chromosomal domains. The NaV1.2 gene is actively transcribed but repressed by REST independently of histone deacetylation or DNA methylation and does not co-localize with epigenetic markers of silence, including dimethylation of H3K9 and HP1. In contrast, the M4 gene is maintained in a silent state independently of REST and co-localizes with dimethylated H3K9 and HP1alpha and HP1gamma, characteristic of silenced or senescent euchromatic DNA. This contrasts with the coordinate REST-dependent regulation of this locus reported previously. Taken together, we infer that distinct repressor complexes and mechanisms are operative at particular loci even in cell lines derived from a common embryological origin.

Animals↗
PubMed2002

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus.

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.

Adenosine Triphosphate↗