Neuronal Transcription Factor Research Articles

Identification of a neuronal transcription factor network involved in medulloblastoma development

BackgroundMedulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development.ResultsMutagenesis significantly increased medulloblastoma frequency and identified 17 candidate cancer genes, including orthologs of genes somatically mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the human disease. Strikingly, these candidate genes were enriched for transcription factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1 and Tgif2) were linked within a single regulatory network enriched for genes associated with a differentiated neuronal phenotype. Furthermore, activity of this network varied significantly between the human subgroups, was associated with metastatic disease, and predicted poor survival specifically within the SHH subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the most differentially expressed gene in murine tumours with network perturbation, and network activity in both mouse and human tumours was characterised by enrichment for multiple gene-sets indicating increased cell proliferation, IGF signalling, MYC target upregulation, and decreased neuronal differentiation.ConclusionsCollectively, our data support a model of medulloblastoma development in SB-mutagenised Ptch+/- mice which involves disruption of a novel transcription factor network leading to Igf2 upregulation, proliferation of GNPs, and tumour formation. Moreover, our results identify rational therapeutic targets for SHH subgroup tumours, alongside prognostic biomarkers for the identification of poor-risk SHH patients.

Functional Analysis of Neuronal MicroRNAs in Caenorhabditis elegans Dauer Formation by Combinational Genetics and Neuronal miRISC Immunoprecipitation

Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic “enhancer” approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.

Prion formation correlates with activation of translation-regulating protein 4E-BP and neuronal transcription factor Elk1

Cellular mechanisms play a role in conversion of the normal prion protein PrPC to the disease-associated protein PrPSc. The cells provide not only PrPC, but also still largely undefined factors required for efficient prion replication. Previously, we have observed that interference with ERK and p38-JNK MAP kinase pathways has opposing effects on the formation of prions indicating that the process is regulated by a balance in intracellualar signaling pathways. In order to obtain a “flow-chart” of such pathways, we here studied the activation of MEK/ERK and mTORC1 downstream targets in relation to PrPSc accumulation in GT1-1 cells infected with the RML or 22L prion strains. We show that inhibition of mTORC1 with rapamycin causes a reduction of PrPSc accumulation at similar low levels as seen when the interaction between the translation initiation factors eIF4E and eIF4G downstream mTORC1 is inhibited using 4EGI-1. No effect is seen following the inhibition of molecules (S6K1 and Mnk1) that links MEK/ERK signaling to mTORC1-mediated control of translation. Instead, stimulation (high [KCl] or [serum]) or inhibition (MEK-inhibitor) of prion formation is associated with increased or decreased phosphorylation of the neuronal transcription factor Elk1, respectively. This study shows that prion formation can be modulated by translational initiating factors, and suggests that MEK/ERK signaling plays a role in the conversion of PrPC to PrPSc via an Elk1-mediated transcriptional control. Altogether, our studies indicate that prion protein conversion is under the control of intracellular signals, which hypothetically, under certain conditions may elicit irreversible responses leading to progressive neurodegenerative diseases.

Neuron and beta‐cell evolution: Learning about neurons is learning about beta‐cells

Neurons and insulin-producing pancreatic beta-cells share multiple common features although they arise from different germ layers 1. In analogy to neurons, beta-cells release insulin by membrane depolarization reminiscent of synaptic neurotransmitter release in neurons and produce the neurotransmitter gamma aminobutyric acid (GABA) in synaptic-like vesicles. Although expression of many individual genes has been shown in neurons and beta-cells, an estimation of the total amount of genes commonly expressed in these cell types has just been addressed recently 2, 3. In a genome-wide mRNA expression scan, profiles of human and rodent beta-cells were compared to non-beta-cell tissues to find genes selectively expressed in beta-cells 2. Interestingly, 15% of these conserved beta-cell markers were also expressed in neuronal tissue coding for proteins involved in neurotransmitter transport, synaptic vesicle formation and brain development 2. To explain why ectodermal-derived neurons and endodermal-derived beta-cells share many common features, Arntfield and van der Kooy have recently speculated that beta-cells may have “borrowed from the brain”, i.e. their ancestors have deployed parts of the neuronal developmental program thereby creating an example of convergent evolution 1. This idea is consistent with the fact that multiple transcription factors are expressed in the neuronal and the pancreatic endocrine lineage including Neurogenin 3 (Ngn3) and NeuroD, both essential for brain and pancreas development. Arntfield et al. have further suggested that the evolution of (modern) beta-cells may have resulted from stochastic effects, i.e. mutation, activating neuronal master transcription factors in gut cells 1. However it remains unclarified which cell type may have first adopted the neuronal program, i.e. a more unspecified gut cell or a pre-differentiated endocrine cell. Recently it has been suggested that beta-cell progenitors co-opt the neural program rather late during their differentiation based on observations on the level of epigenetics 3. Interestingly, neuronal genes are still repressed in multipotent pancreatic progenitors that can give rise to exo- or endocrine tissue suggesting that an activation of neuronal gene activity occured later in the endocrine lineage 3. This would indicate that a pre-differentiated multipotent pancreatic progenitor adopted the neuronal program rather than a more unspecified cell. The notion that neurons and beta-cells are similar has stimulated scientists to transfer principles from neurobiology to understand beta-cell function and to find drugs to support beta-cell function under stress conditions as observed in type 2 diabetes mellitus. For example, screening of a chemical library for neurogenic activators has recently led to the identification of a potent inducer of NeuroD, supporting insulin production in pancreatic beta-cells 4. Moreover, known neuroprotective mechanisms may also be essential in beta-cells as observed in the case of the Parkinson-related antioxidant protein DJ-1 5. DJ-1 preserves mitochondrial integrity in dopaminergic neurons that are vulnerable to reactive oxygen species. In analogy to neurons, DJ-1 also protects the mitochondrial function of beta-cells suggesting that beta-cells and neurons share similar oxidative stress defence mechanisms. Thus, the study of common principles in neurons and beta-cells may provide a basis for prevention and treatment of diabetes mellitus and neurodegenerative diseases.

Strong expression of the neuronal transcription factor FOXP2 is linked to an increased risk of early PSA recurrence in ERG fusion-negative cancers

Background and aimsTranscription factors of the forkhead box P (FOXP1-4) family have been implicated in various human cancer types before. The relevance and role of neuronal transcription factor FOXP2 in...

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy - The Turning Point of Cell-Based Regenerative Medicine.

To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point.

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine

To date, the lack of a clinically-suitable source of engraftable human stem/progenitor cells with adequate neurogenic potential has been the major setback in developing safe and effective cell-based therapies for regenerating the damaged or lost CNS structure and circuitry in a wide range of neurological disorders. Similarly, the lack of a clinically-suitable human cardiomyocyte source with adequate myocardium regenerative potential has been the major setback in regenerating the damaged human heart. Given the limited capacity of the CNS and heart for self-repair, there is a large unmet healthcare need to develop stem cell therapies to provide optimal regeneration and reconstruction treatment options to restore normal tissues and function. Derivation of human embryonic stem cells (hESCs) provides a powerful in vitro model system to investigate molecular controls in human embryogenesis as well as an unlimited source to generate the diversity of human somatic cell types for regenerative medicine. However, realizing the developmental and therapeutic potential of hESC derivatives has been hindered by the inefficiency and instability of generating clinically-relevant functional cells from pluripotent cells through conventional uncontrollable and incomplete multi-lineage differentiation. Recent advances and breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including establishing defined culture systems for de novo derivation and maintenance of clinical-grade pluripotent hESCs and lineage-specific differentiation of pluripotent hESCs by small molecule induction. Retinoic acid was identified as sufficient to induce the specification of neuroectoderm direct from the pluripotent state of hESCs and trigger a cascade of neuronal lineage-specific progression to human neuronal progenitors and neurons of the developing CNS in high efficiency, purity, and neuronal lineage specificity by promoting nuclear translocation of the neuronal specific transcription factor Nurr-1. Similarly, nicotinamide was rendered sufficient to induce the specification of cardiomesoderm direct from the pluripotent state of hESCs by promoting the expression of the earliest cardiac-specific transcription factor Csx/Nkx2.5 and triggering progression to cardiac precursors and beating cardiomyocytes with high efficiency. This technology breakthrough enables direct conversion of pluripotent hESCs into a large supply of high purity neuronal cells or heart muscle cells with adequate capacity to regenerate CNS neurons and contractile heart muscles for developing safe and effective stem cell therapies. Transforming pluripotent hESCs into fate-restricted therapy derivatives dramatically increases the clinical efficacy of graft-dependent repair and safety of hESC-derived cellular products. Such milestone advances and medical innovations in hESC research allow generation of a large supply of clinical-grade hESC therapy derivatives targeting for major health problems, bringing cell-based regenerative medicine to a turning point.

Do we have to reconsider the evolutionary emergence of myelin?

Do we have to reconsider the evolutionary emergence of myelin?

Npas4: A Neuronal Transcription Factor with a Key Role in Social and Cognitive Functions Relevant to Developmental Disorders

Npas4 is a transcription factor, which is highly expressed in the brain and regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity. A deregulation of the inhibitory-excitatory balance has been associated with a variety of human developmental disorders such as schizophrenia and autism. However, not much is known about the role played by inhibitory synapses and inhibitory pathways in the development of nervous system disorders. We hypothesized that alterations in the inhibitory pathways induced by the absence of Npas4 play a major role in the expression of the symptoms observed in psychiatric disorders. To test this hypothesis we tested mice lacking the transcription factor (Npas4 knock-out mice (Npas4-KO)) in a battery of behavioral assays focusing on general activity, social behaviors, and cognitive functions. Npas4-KO mice are hyperactive in a novel environment, spend less time exploring an unfamiliar ovariectomized female, spend more time avoiding an unfamiliar male during a first encounter, show higher social dominance than their WT littermates, and display pre-pulse inhibition, working memory, long-term memory, and cognitive flexibility deficits. These behavioral deficits may replicate schizophrenia-related symptomatology such as social anxiety, hyperactivity, and cognitive and sensorimotor gating deficits. Immunohistochemistry analyses revealed that Npas4 expression is induced in the hippocampus after a social encounter and that Npas4 regulates the expression of c-Fos in the CA1 and CA3 regions of the hippocampus after a cognitive task. Our results suggest that Npas4 may play a major role in the regulation of cognitive and social functions in the brain with possible implications for developmental disorders such as schizophrenia and autism.

Trophic factors differentiate dopamine neurons vulnerable to Parkinson's disease

Recent studies suggest a variety of factors characterize substantia nigra neurons vulnerable to Parkinson's disease, including the transcription factors pituitary homeobox 3 (Pitx3) and orthodenticle homeobox 2 (Otx2) and the trophic factor receptor deleted in colorectal cancer (DCC), but there is limited information on their expression and localization in adult humans. Pitx3, Otx2, and DCC were immunohistochemically localized in the upper brainstem of adult humans and mice and protein expression assessed using relative intensity measures and online microarray data. Pitx3 was present and highly expressed in most dopamine neurons. Surprisingly, in our elderly subjects no Otx2 immunoreactivity was detected in dopamine neurons, although Otx2 gene expression was found in younger cases. Enhanced DCC gene expression occurred in the substantia nigra, and higher amounts of DCC protein characterized vulnerable ventral nigral dopamine neurons. Our data show that, at the age when Parkinson's disease typically occurs, there are no significant differences in the expression of transcription factors in brainstem dopamine neurons, but those most vulnerable to Parkinson's disease rely more on the trophic factor receptor DCC than other brainstem dopamine neurons.

The neurogenic basic helix-loop-helix transcription factor NeuroD6 enhances mitochondrial biogenesis and bioenergetics to confer tolerance of neuronal PC12-NeuroD6 cells to the mitochondrial stressor rotenone

The fundamental question of how and which neuronal specific transcription factors tailor mitochondrial biogenesis and bioenergetics to the need of developing neuronal cells has remained largely unexplored. In this study, we report that the neurogenic basic helix-loop-helix transcription factor NeuroD6 possesses mitochondrial biogenic properties by amplifying the mitochondrial DNA content and TFAM expression levels, a key regulator for mitochondrial biogenesis. NeuroD6-mediated increase in mitochondrial biogenesis in the neuronal progenitor-like PC12-NEUROD6 cells is concomitant with enhanced mitochondrial bioenergetic functions, including increased expression levels of specific subunits of respiratory complexes of the electron transport chain, elevated mitochondrial membrane potential and ATP levels produced by oxidative phosphorylation. Thus, NeuroD6 augments the bioenergetic capacity of PC12-NEUROD6 cells to generate an energetic reserve, which confers tolerance to the mitochondrial stressor, rotenone. We found that NeuroD6 induces an adaptive bioenergetic response throughout rotenone treatment involving maintenance of the mitochondrial membrane potential and ATP levels in conjunction with preservation of the actin network. In conclusion, our results support the concept that NeuroD6 plays an integrative role in regulating and coordinating the onset of neuronal differentiation with acquisition of adequate mitochondrial mass and energetic capacity to ensure energy demanding events, such as cytoskeletal remodeling, plasmalemmal expansion, and growth cone formation.

NRSF/REST neuronal deficient mice are more vulnerable to the neurotoxin MPTP

Parkinson's disease (PD) is characterized by progressing loss of dopaminergic neurons in the midbrain. Abnormal gene expression plays a critical role in its pathogenesis. Neuron-restrictive silencer factor (NRSF)/neuronal repressor element-1 silencing transcription factor (REST), a member of the zinc finger transcription factors, inhibits the expression of neuron-specific genes in nonneuronal cells, and regulates neurogenesis. Our previous work showed that 1-methyl-4-phenyl-pyridinium ion triggers dynamic changes of messenger RNA and protein expression of NRSF in human dopaminergic SH-SY5Y cells, and alteration of NRSF expression exacerbates 1-methyl-4-phenyl-pyridinium ion-induced cell death. The purpose of this study was to explore the in vivo role of NRSF in the progress of PD by using NRSF/REST neuron-specific conditional knockout mice (cKO). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was adopted to generate PD models in the cKO mice and wild type littermates. At 1, 3, 7, 14, 21, and 28 days after MPTP injection, behavioral tests were performed, and cKO mice displayed some impairments in locomotor activities. Also, the reduction of tyrosine hydroxylase protein in the striatum and the loss of dopaminergic neurons in the substantia nigra were more severe in the cKO mice. Meanwhile, the cKO mice exhibited a more dramatic depletion of striatal dopamine, accompanied by an increase in glial fibrillary acidic protein (GFAP) expression and sustained interleukin-1β transcription. These results suggested that NRSF/REST neuronal cKO mice are more vulnerable to the dopaminergic neurotoxin MPTP. Disturbance of the homeostasis of NRSF and its target genes, gliogenesis, and inflammation may contribute to the higher MPTP sensitivity in NRSF/REST neuronal cKO mice.

Expression of P450c17 in the Human Fetal Nervous System

P450c17 catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. P450c17 is expressed in human fetal and postnatal adrenals and gonads and in the developing mouse nervous system, but little is known about its expression in the human nervous system. We obtained portions of 9-, 10-, and 11-wk gestation human fetuses and delineated the pattern of expression of P450c17 in their peripheral nervous systems by immunocytochemistry using the P450c17 antiserum previously used to characterize P450c17 in the mouse brain. P450c17 was readily detected in the dorsal root ganglia (DRG) and spinal cord. Neural structures were identified with antisera to the cytoskeletal protein neural cell adhesion molecule; DRG were identified with antisera to the neuronal transcription factor BRN3A and neurotrophin receptor tropomyosin-receptor-kinase B. The identification of P450c17 was confirmed using commercial antisera directed against different domains of P450c17 and by using antisera immunodepleted with authentic human P450c17. We also found expression of the P450 cholesterol side-chain cleavage enzyme (P450scc) in the spinal cord and DRG. Expression of P450scc is limited to cell bodies; unlike P450c17, we never detected P450scc in fiber tracts. Catalysis by P450c17 requires electron donation from P450 oxidoreductase (POR). Dual-label immunohistochemistry detected P450c17 and POR colocalized in DRG bundles, but some fibers containing P450c17 lacked POR. These data suggest that neurosteroids synthesized via these two enzymes may act in the developing human nervous system. The expression of P450c17 in structures lacking POR means that P450c17 may not be steroidogenic in those locations, suggesting that P450c17 may have additional functions that do not require POR.

In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors

In vitro exposure of neural progenitor cell (NPC) populations to reduced O2 (e.g. 3% versus 20%) can increase their proliferation, survival and neuronal differentiation. Our objective was to determine if an acute (<1hr), in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20×1 minute; alternating 21% and 10% O2). Immediately after AIH, SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC, and this was accompanied by increases in a proliferation maker (Ki67), MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally, immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue, and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to “prime” NPC prior to transplantation into the injured central nervous system.

Nonviral Direct Conversion of Primary Mouse Embryonic Fibroblasts to Neuronal Cells

Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm−2 doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1+ (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP+ cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.

CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease.

Nontelomeric splice variant of telomere repeat-binding factor 2 maintains neuronal traits by sequestering repressor element 1-silencing transcription factor

Telomere repeat-binding factor 2 (TRF2) is critical for telomere integrity in dividing stem and somatic cells, but its role in postmitotic neurons is unknown. Apart from protecting telomeres, nuclear TRF2 interacts with the master neuronal gene-silencer repressor element 1-silencing transcription factor (REST), and disruption of this interaction induces neuronal differentiation. Here we report a developmental switch from the expression of TRF2 in proliferating neural progenitor cells to expression of a unique short nontelomeric isoform of TRF2 (TRF2-S) as neurons establish a fully differentiated state. Unlike nuclear TRF2, which enhances REST-mediated gene repression, TRF2-S is located in the cytoplasm where it sequesters REST, thereby maintaining the expression of neuronal genes, including those encoding glutamate receptors, cell adhesion, and neurofilament proteins. In neurons, TRF2-S-mediated antagonism of REST nuclear activity is greatly attenuated by either overexpression of TRF2 or administration of the excitatory amino acid kainic acid. Overexpression of TRF2-S rescues kainic acid-induced REST nuclear accumulation and its gene-silencing effects. Thus, TRF2-S acts as part of a unique developmentally regulated molecular switch that plays critical roles in the maintenance and plasticity of neurons.

Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo

Reprogramming factors have been used to induce pluripotent stem cells as an alternative to somatic cell nuclear transfer technology in studies targeting disease models and regenerative medicine. The neuronal repressor RE-1 silencing transcription factor (REST) maintains self-renewal and pluripotency in mouse embryonic stem cells by maintaining the expression of Oct3/4, Nanog, and cMyc. We report that primary hepatocytes express REST and most of the reprogramming factors in culture. Their expression is up-regulated by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). REST inhibition results in down-regulation of reprogramming factor expression, increased apoptosis, decreased proliferation, and cell death. The reprogramming factors are also up-regulated after 70% partial hepatectomy in vivo. These findings show that genes inducing the iPS phenotype, even though expressed at lower levels than embryonic stem cells, nonetheless are associated with control of apoptosis and cell proliferation in hepatocytes in culture and may play a role in such processes during liver regeneration.

Clinically relevant single gene or intragenic deletions encompassing critical neurodevelopmental genes in patients with developmental delay, mental retardation, and/or autism spectrum disorders

Recent studies suggest that copy number variations (CNVs) encompassing several genes involved in neurodevelopmental pathways are associated with a variety of neuropsychiatric phenotypes, including developmental delay (DD), mental retardation (MR), and autism spectrum disorders (ASDs). Here we present eight patients in a cohort of approximately 1,200 patients referred for clinical array CGH testing for various neurodevelopmental phenotypes,whowere identified to carry small (<1.0Mb with the majority <500 kb) either total gene or intragenic deletions encompassing critical synaptic and other neurodevelopmental genes. The presentations of these patients included variable degrees of DD, speech problems, learning disabilities, MR, autistic-like features, and mild non-specific dysmorphic features. These genes belong to four functional categories, including neuronal transcription factor genes (NFIA at 1p31.3, MEF2C at 5q14.3, andCAMAT1at 1p36.23p36.31), neuron-specific splicing factor genes (RBFOX1 at 16p13.2p13.3), genes involved in synapse formation and maintenance (CNTNAP2 at 7q35 and LRFN5 at 14q21.2), and genes involved in neurotransmission (CHRNA7 at 15q13.3 and IL1RAPL1 at Xp21.2p21.3). Our report expands the list of neurodevelopmental genes deleted in various neurobehavioral phenotypes, expands the phenotypes caused by haploinsufficiency of previously reported critical neurodevelopmental genes, and elucidates the clinical relevance and need for careful clinical interpretation of some small CNVs<500 kb. This report also suggests that small clinically relevant deletions encompassing critical synaptic and other neurodevelopmental genes can present clinically with various neurobehavioral phenotypes, which implies the existence of overlapping neuronal pathways in the pathogenesis of these phenotypes.

In Vivo Neuronal Subtype-Specific Targets of Atoh1 (Math1) in Dorsal Spinal Cord

Neural basic helix-loop-helix (bHLH) transcription factors are crucial in regulating the differentiation and neuronal subtype specification of neurons. Precisely how these transcription factors direct such processes is largely unknown due to the lack of bona fide targets in vivo. Genetic evidence suggests that bHLH factors have shared targets in their common differentiation role, but unique targets with respect to their distinct roles in neuronal subtype specification. However, whether neuronal subtype-specific targets exist remains an unsolved question. To address this question, we focused on Atoh1 (Math1), a bHLH transcription factor that specifies distinct neuronal subtypes of the proprioceptive pathway in mammals including the dI1 (dorsal interneuron 1) population of the developing spinal cord. We identified transcripts unique to the Atoh1-derived lineage using microarray analyses of specific bHLH-sorted populations from mouse. Chromatin immunoprecipitation-sequencing experiments followed by enhancer reporter analyses identified five direct neuronal subtype-specific targets of Atoh1 in vivo along with their Atoh1-responsive enhancers. These targets, Klf7, Rab15, Rassf4, Selm, and Smad7, have diverse functions that range from transcription factors to regulators of endocytosis and signaling pathways. Only Rab15 and Selm are expressed across several different Atoh1-specified neuronal subtypes including external granule cells (external granule cell layer) in the developing cerebellum, hair cells of the inner ear, and Merkel cells. Our work establishes on a molecular level that neuronal differentiation bHLH transcription factors have distinct lineage-specific targets.