Toward synaptic transcriptomes: Direct sequencing and identification of RNAs actively transported by the kinesin complex from the cell body to synapses in Aplysia neurons
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ABSTRACT: Specific mRNAs are transported from the cell body to synapses where their translation can modify communication of pre-existing synapses and induce formation of new synaptic connections in response to learning. Little is known, however, about the identity of the RNAs that are actively transported and when and how these RNAs are utilized during learning. By focusing on RNAs that are associated with kinesin, a motor protein that transports gene products from the cell body to synapses, we have now applied microarrays and 454 sequencing to identify actively transported RNAs from the Aplysia central nervous system. Using a library prepared from the kinesin complex immunoprecipitated from the central nervous system (CNS), we have identified thousands of unique transcripts, of which ~600 mRNAs were annotated. Two sample comparison: kinesin IP vs. control.
Project description:The complexity of events associated with age-related memory loss (ARML) cannot be overestimated. The problem is further complicated by the enormous diversity of neurons in the CNS and even synapses of one neuron within a neural circuit. Large-scale single-neuron analysis is not only challenging but mostly impractical for any model currently used in ARML. We simply do not know: do all neurons and synapses age differently or are some neurons (or synapses) more resistant to aging than others? What is happening in any given neuron while it undergoes “normal” aging? What are the genomic changes that make aging apparently irreversible? What would be the balance between neuron-specific vs global genome-wide changes in aging? In the proposed paper we address these questions and develop a new model to study the entire scope of genomic and epigenomic regulation in aging at the resolution of single functionally characterized cells and even cell compartments. In particular, the mollusc Aplysia californica has been implemented as a powerful paradigm in addressing fundamental questions of the neurobiology of aging. The proposed manuscript will consist of four parts. First, we will provide an introduction to Aplysia as a representative of the largest superclade of bilaterian animals (Lophotrochozoa). Aplysia has a short lifespan of 220-300 days with a well-characterized life cycle and characterized phenomenology of aging. Most importantly, Aplysia possess the largest nerve cells in the entire animal kingdom (only eggs are larger); these cells can be uniquely identified and mapped in terms of their well-defined interactions with other neurons forming relatively simpler neural circuits underlying several stereotypic and learned behaviors. Second, we have identified in Aplysia more that a hundred neurological- and age-related genes that were lost in other established invertebrate models (such as Drosophila and C. elegans). The proposed long-term regulatory age-related mechanisms include a high level of conservation among many epigenetic processes known to be lost in nematodes and flies with extremely short lifecycles and particularly derived genomes. We also identify and cloned more than 30 evolutionarily conserved homologs of genes involved in Alzheimer’s, Parkinson’s and Huntington’s diseases as well as age-related hormones. Third, we performed genome-wide analysis of expression patterns of more than 55,000 unique transcripts by comparing two different identified cholinergic neurons (R2 and LPl1) among young and aged animals. This direct single neuron genomic analysis indicates that there are significant cell-specific changes in gene-expression profiles as a function of aging. We estimated that only ~10-20% of genes that are differently expressed in the aging brain are common for all neuronal types - the remaining 80% are neuron-specific (i.e. found in aging neurons of one but not another type). The list of “common aging genes” includes components of insulin growth factor pathways, cell bioenergetics, telomerase-associated proteins, antioxidant enzymes, water channels and estrogen receptors. The rest were neuron-specific gene products (including apoptosis-related proteins, Alzheimer-related genes, growth factors and their receptors, ionic channels, transcription factors and more than 120 identified proteins known to be involved in neurodevelopment and synaptogenesis). Surprisingly, even two different identified cholinergic motoneurons age differently and each of them has a unique subset of genes differentially expressed in older animals. Fourth, we showed that the activity of the entire genome and associated epigenomic modifications (e.g. DNA methylation, histone dynamics) can be efficiently monitored within a single Aplysia neuron and can be modified as a function of aging in a neuron-specific manner including selective histones and histone-modifying enzymes and DNA methylation-related enzymes. This genome-wide analysis of aging allows us to propose novel mechanisms of active DNA demethylation and cell-specific methylation as well as regional relocation of RNAs as three key processes underlying age-related memory loss. These mechanisms tune the dynamics of long-term chromatin remodeling, control weakening and the loss of synaptic connections in aging. At the same time, our genomic tests revealed evolutionarily conserved gene clusters in the Aplysia genome associated with senescence and regeneration (e.g. apoptosis- and redox- dependent processes, insulin signaling, etc.). This is a reference design experiment with all samples being compared to one CNS from Aplysia. Two cholinergic neurons (R2 and LPl1), two ages (young and old), two arrays (AAA and DAA), three biological replicates each sample type. Two direct comparison experiments were also performed. One with young and old abdominal ganglion and the other with young and old R2.
Project description:Specific mRNAs are transported from the cell body to synapses where their translation can modify communication of pre-existing synapses and induce formation of new synaptic connections in response to learning. Little is known, however, about the identity of the RNAs that are actively transported and when and how these RNAs are utilized during learning. By focusing on RNAs that are associated with kinesin, a motor protein that transports gene products from the cell body to synapses, we have now applied microarrays and 454 sequencing to identify actively transported RNAs from the Aplysia central nervous system. Using a library prepared from the kinesin complex immunoprecipitated from the central nervous system (CNS), we have identified thousands of unique transcripts, of which ~600 mRNAs were annotated.
Project description:The aim of the study was to find novel genes highly upregulated in mouse mucosal mast cells. To carry it out, differentiating progenitor cells were isolated on day4 of culturing and compared to mature mucosal mast cells obtained by day20. Experiment Overall Design: The experiment was carried out on two microarrays. Array2 is a biological and technical replicate (dye-swap) of array1. In both cases, 3-3 samples were pooled, but of different origin.
Project description:Whole genome transcriptional profiling is used to identify ESTs found in RNA co-immunoprecipitated from Aplysia CNS with antibodies against Aplysia Kinesin Heavy Chain (ApKHC) RNA derived from Aplysia CNS served as control in the opposite channel. Custom Aplysia EST array to probe the samples was designed and ordered from Agilent. Two-condition experiment; four biological replicates for each condition were reciprocally hybridized on each two-color array
Project description:Toward synaptic transcriptomes: Direct sequencing and identification of RNAs actively transported by the kinesin complex from the cell body to synapses in Aplysia neurons
Project description:Understanding cell-type-specific epigenetic codes on a global level is a major challenge after the sequencing of the human genome has been completed. Here we applied methyl-CpG immunoprecipitation (MCIp) to obtain comparative methylation profiles of coding and noncoding genes in three human tissues, testis, brain, and monocytes. Forty-four mainly testis-specific promoters were independently validated using bisulfite sequencing or single-gene MCIp, confirming the results obtained by the MCIp microarray approach. We demonstrate the previously unknown somatic hypermethylation at many CpG-rich, testis-specific gene promoters, in particular in ampliconic areas of the Y chromosome. We also identify a number of miRNA genes showing tissue-specific methylation patterns. The comparison of the obtained tissue methylation profiles with corresponding gene expression data indicates a significant association between tissue-specific promoter methylation and gene expression, not only in CpG-rich promoters. In summary, our study highlights the exceptional epigenetic status of germ-line cells in testis and provides a global insight into tissue-specific DNA methylation patterns. Keywords: MCIp-on-Chip The promoter hypomethylation profiles of the two somatic tissues (monocytes and brain) were compared to human testis (reference). The set includes two hybridisations with independent testis samples for each comparison. Each comparison uses a set of two microarrays.
Project description:The monitoring of global transcription patterns during development is a useful first step to understand mechanisms underlying growth, differentiation and patterning in a given species. However such large scale developmental studies are so far only available from a few selected model organisms such as mouse, Drosophila and the nematode C. elegans. Genomic scale information from the lophotrochozoa is now emerging. The recently characterized neuronal transcriptome of the sea hare Aplysia californica (~200’000 ESTs and ~40,000 unique non-redundant sequences representing about 55-70% of all neuronal transcripts including splice forms and non-coding RNAs) provides new insights and opportunities into problems of molluscan development and specifically neurogenesis. Regulatory genes used in development are recruited for the functions of adult nervous systems such as synaptogenesis, specific wiring in networks and structural changes underlying synaptic plasticity. While this similarity reflects a basic concept in biology, namely the recruitment of the same genes for different functions, it has never been tested on a large scale genomic level. We used representative oligo-arrays constructed from transcripts obtained from the Aplysia nervous system to explore the following two questions: 1) What neuronal genes are differentially expressed during embryonic development of the sea hare Aplysia. 2) What gene expression changes can be observed in correlation with gastrulation, metamorphosis, and larval neurogenesis. For this purpose we hybridized RNA from 3 embryonic (64 cells, gastrula, trochophore) and 4 larval stages (pre-hatching, post-hatching, pre-metamorphosis, postmetamorphosis) against a reference sample consisting of equal contributions from all stages. Our analysis revealed that all embryonic and larval stages can be distinguished based on their transcription profiles. A comparison of the Aplysia atlas with similar studies in Drosophila and mouse reveals interesting differences. We identified several new transcription factors which are differentially expressed during early embryonic development. Various other transcripts involved in metabolism and differentiation were characterized. These genes can be candidate targets for understanding neuronal growth, synaptogenesis and memory mechanisms. We conclude that the microarray technology provides us with a powerful tool for efficient survey and functional annotation of the neuronal transcriptome in Aplysia. We used a reference design for this study where we hybridized each of the 8 developmental stages of Aplysia (cleavage, gastrula, trochophore, first veliger, hatching veliger, pre-metamorphosis (stage 6), post-metamorphosis (stage 7), post-metamorphosis 60 hours (pm60)
Project description:The most widely-used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms and analysis algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. All commercial tiling array platforms performed well, although each platform and analysis algorithm had distinct performance and cost characteristics. Simple sequence repeats and genome redundancy tend to result in false positives on oligonucleotide platforms. The spike-in DNA samples and the resulting array data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: chip-ChIP simulation For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf For each replicate array, we labeled 1µg of spike-in sample and 1µg of control sample. The Bioprime Plus Array CGH labeling Module (Invitrogen Catolog number 18095-014) was used. The spike-in was labeled with the red channel dye (Alexa Fluor-647) and the control was labeled with the green channel dye (Alexa Fluor-555) in two of the replicates. A dye swap was performed for the third replicate. These samples were then competitively hybridized to three replicate 244k arrays from Agilent, (AMADID 25150451). We hybridized the samples to the arrays using the Agilent Array CGH protocol, with some modifications. Briefly, labeled DNA was combined with Cot-1 DNA, blocking reagent, and hybridization buffer. The hybridizations were carried out at 60°C. The arrays were scanned using an Agilent dual laser scanner, and the images were processed using Agilent Feature Extraction Software.
Project description:This series consists of samples taken from two groups of K562 cells(miR-181a transfected group and control group ) and harvested 48 hours later. We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Further studies aimed to find target mRNA of miR-181a in mammalian cells and its biological function. Experiment Overall Design: The K562 cells in our research were divided into tranfected group and mock-transfected control according to Lim et al. Experiment Overall Design: To transfect, we diluted 60 pmol miR-181 a duplex into 12 µL Opti-Mem each well, then we diluted 3 µL Oligofectamine(Invitrogen) into 48 µL Opti-Mem for each sample, incubated for 5 min at room temperature and add 10 µL of diluted transfection mixture to wells and incubate for another 20 min at room temperature. We next added 100 µL of diluted cells suspension mixture containing 50000 cells on top of the complex. Four hours after transfection, FBS was added to the cells at a final concentration of 10% and the cells were incubated for 48 hours. And control group was transfected only with Oligofectamine but no RNA. Each individual transfection experiment was performed in 10 replicate wells. Experiment Overall Design: After transfection experiments, total RNAs from above two group K562 cells were purified using the RNeasy mini kit (Qiagen, Heidelberg, Germany) in accordance with the manufacturerâs protocols. RNA concentration and purity were estimated spectrophotometrically and the quality further assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Experiment Overall Design: Total RNA from control group and transfected group were respectively reverse transcription, in vitro transcribed and labeled (Cy3 and Cy5) according to the Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol, green is control group, red is transfected group. The array hybridization, washing, and scanning procedures were performed in our lab according to Agilent 60-mer oligo microarray processing protocol using 0.75 µg samples of labeled, fragmented cRNA on Agilent Human 1A oligo microarray with 22575 probes (Agilent Technologies). Labeled cRNA is incubated with the microarray in a hybridization chamber at 60ºC for 17 hours, 4 rpm/min. The microarray was scanned using Agilent 2565BA DNA microarray scanner (Agilent Technologies). Data collection was performed by Agilent DNA microarray scanner. Using the Agilent Feature Extraction Software(v.7.5), spots were identified, measured for fluorescence intensity, local background, and arraywide parameters used to normalize the signals. Outliers and saturated spots were rejected according to 1.42xIQR, and local backgroud subtracted from the signals. Signals were then normalized to remove dye bias and arraywide drift in flourescence. Experiment Overall Design: The following two tests are performed to determined the significance of a feature: (1) Log ratio P-value of the red over green processed signals was calculated using two-sided t-test with P<0.01 considered significant, and (2) a boolean flag of 1 indicated that the feature BGSubSignal is well above background and passes the IsPosAndSignif test. Experiment Overall Design: We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Experiment Overall Design: Further studies aimed to find target mRNA of miR-181a in mammalian cells and the function of miR-181a in cancers.
Project description:The Infectious Hematopoietic Necrosic Virus (IHNV) DNA vaccine is based on the viral glycoprotein gene (G gene) and induces a non-specific anti-viral immune response and long-term specific immunity against IHNV in salmonid fishes. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression at the I.M. site was evaluated using a 16,000 feature salmon cDNA microarray. Eighty different transcripts were significantly modulated in the vector DNA group while 910 transcripts were modulated in the IHNV DNA vaccinated group relative to control group. Quantitative reverse transcriptase PCR was used to examine expression of selected genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were higher in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen) evaluated with RT-PCR of specific genes, the main difference was the up-regulation of the type I IFN-related genes in only the IHNV DNA vaccinated group. Keywords: disease vaccine response Gene expression at the site of I.M injection was evaluated 7 days post-injection in clonal rainbow trout. Gene expression was compared across three treatments, with replicate clonal rainbow trout individuals (Hot Creek strain) within each treatment. Treatments included injection with 1X PBS (control group), plasmid vector only (without IHNV G gene; hereafter referred to as vector group), and the pIHNV vaccine (vaccine group). Three clonal individuals from the control, three individuals from the vector group, and four individuals from the vaccine group were labeled with Cy3 and used for individual hybridizations to ten slides, comparing hybridization of each individual to a major reference RNA pool. The major reference pool consisted of three unhandled clonal rainbow trout and constituted a pool of RNA from the anterior kidney, spleen, liver and muscle and was labelled with Cy5. Total RNA was extracted from samples, amplified, and hybridized to 16,000 feature Atlantic salmon cDNA arrays developed by the Genomic Research for Atlantic Salmon project. Differential expression was compared among control, vector, and vaccine groups.