La correlación de Gene-específica de ADN de metilación cambios con la expresión y actividad transcripcional de astrocíticos<em> KCNJ10</em> (Kir4.1)
Summary
DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.
Abstract
DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.
Introduction
Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2.
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.
Protocol
Representative Results
Discussion
This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge i…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.
Materials
| Papain Dissociation System | Worthington Biochemical Corporation | LK003150 | |
| AllPrep DNA/RNA Mini Kit | Qiagen | 80204 | |
| Methyl Primer | Applied Biosystems | online | software to localize CpG Islands |
| EZ DNA methylation Kit | Zymo Research | D5001 | |
| Rat Premixed Calibration Standard | EpiGenDx | 80-8060R-Premix | |
| CpG Methylase (M.Sssl) | Zymo Research | E2010 | |
| QIAquick Gel Extraction | QIagen | 28704 | Used for gel extraction and DNA cleanup |
| Restriction enzymes | New England BioLabs | ||
| NEB cutter | New England BioLabs | online | verify restriction digest sites |
| Dual Luciferase Reporter Assay System | Promega | E1910 | |
| Luc2 vector, pGL4.10 | Promega | E6651 | |
| renilla vector, pGL4.74 | Promega | E2241 | |
| TD-20/20 Luminometer | Turner Designs | ||
| Lipofectamine LTX and Plus Reagent | Life Technologies | A12621 | |
| Phenol, saturated pH 6.6/6.9 | Fisher Scientific | BP 17501-100 | |
| Nanodrop 2000/2000c Spectrophtometer | ThermoScientific | ||
| MeltDoctor Master Mix | Life Technologies | 4415440 | |
| High Resolution Melt (HRM) Software v2.0 | Life Technologies | 4397808 | |
| AB SDS software v2.3 | Life Technologies | online | |
| AB High Resolution Melting Getting Started Guide | Life Technologies | online | |
| AB 7900HT Fast Real-Time System | Life Technologies |
Riferimenti
- Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16 (1), 6-21 (2002).
- Deaton, A. M., Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010-1022 (2011).
- Lorincz, M. C., Dickerson, D. R., Schmitt, M. Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nat Struct. Mol Biol. 11 (11), 1068-1075 (2004).
- Moore, L. D., Le, T. DNA methylation and its basic function. Neuropsychopharmacol. 38, 23-38 (2013).
- Santos, K. F., Mazzola, T. N. The prima donna of epigenetics: the regulation of gene expression by DNA methylation. Braz.J.Med.Biol.Res. 38 (10), 1531-1541 (2005).
- Day, J. J., Sweatt, J. D. Cognitive neuroepigenetics: a role for epigenetic mechanisms in learning and memory. Neurobiol. Learn.Mem. 96, 2-12 (2011).
- Denk, F., McMahon, S. B. Chronic pain: emerging evidence for the involvement of epigenetics. Neuron. 73, 435-444 (2012).
- Endres, M., et al. DNA methyltransferase contributes to delayed ischemic brain injury. J. Neuroscience. 20 (9), 3175-3181 (2000).
- Qureshi, I. A., Mehler, M. F. Emerging role of epigenetics in stroke: part 1: DNA methylation and chromatin modifications. Arch.Neurol. 67, 1316-1322 (2010).
- Tian, R., et al. disease mutant glial fibrillary acidic protein compromises glutamate transport in astrocytes. J Neuropathol. Exp Neurol. 69, 335-345 (2010).
- Perisic, T., Holsboer, F., Rein, T. The CpG island shore of the GLT-1 gene acts as a methylation-sensitive enhancer. Glia. 60 (9), 1345-1355 (2012).
- Olsen, M. L., Higashimori, H., Campbell, S. L., Hablitz, J. J. Functional expression of Kir4.1 channels in spinal cord astrocytes. Glia. 53 (5), 516-528 (2006).
- Poopalasundaram, S., et al. Glial heterogeneity in expression of the inwardly rectifying K+ channel, Kir4.1, in adult rat CNS. Glia. 30, 362-372 (2000).
- Hibino, H., Fujita, A., Iwai, K., Yamada, M. Differential assembly of inwardly rectifying K+ channel subunits. Kir4.1 and Kir5.1, in brain astrocytes. 279, 44065-44073 (2004).
- Nwaobi, S. E., Lin, E., Peramsetty, S. R. DNA methylation functions as a critical regulator of Kir4.1 expression during CNS development. Glia. 62 (3), 411-427 (2014).
- Li, L., Head, V. Identification of an inward rectifier potassium channel gene expressed in mouse cortical astrocytes. Glia. 33, 57-71 (2001).
- Kucheryavykh, Y. V., et al. Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes. Glia. 55 (3), 274-281 (2007).
- Djukic, B., Casper, K. B., Philpot, B. D., Chin, L. S. Conditional knock-out of Kir4.1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation. J. Neuroscience. 27, 11354-11365 (2007).
- Fujita, A., et al. Clustering of Kir4.1 at specialized compartments of the lateral membrane in ependymal cells of rat brain. Cell Tissue Res. 359 (2), 627-634 (2015).
- MacFarlane, S. N., Sontheimer, H. Electrophysiological changes that accompany reactive gliosis in vitro. J Neuroscience. 17 (19), 7316-7329 (1997).
- Ambrosio, R., Maris, D. O., Grady, M. S., Winn, H. R. Impaired K(+) homeostasis and altered electrophysiological properties of post-traumatic hippocampal glia. J. Neuroscience. 19 (18), 8152-8162 (1999).
- Anderova, M., et al. Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound. Glia. 48 (4), 311-326 (2004).
- Olsen, M. L., Campbell, S. C., McFerrin, M. B., Floyd, C. L. Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17 beta-oestradiol treatment. Brain. 133, 1013-1025 (2010).
- Koller, H., Schroeter, M., Jander, S., Stoll, G. Time course of inwardly rectifying K(+) current reduction in glial cells surrounding ischemic brain lesions. Brain Res. 872, 1-2 (2000).
- Bordey, A., Lyons, S. A., Hablitz, J. J. Electrophysiological characteristics of reactive astrocytes in experimental cortical dysplasia. J Neurophysiol. 85 (4), 1719-1731 (2001).
- Albuquerque, C., Joseph, D. J., Choudhury, P. . plating, and maintenance of cortical astrocyte cultures. 8, 10-1101 (2009).
- Itakura, E., et al. Generation of transgenic rats expressing green fluorescent protein in S-100beta-producing pituitary folliculo-stellate cells and brain astrocytes. Endocrinology. 148, 1518-1523 (2007).
- Foo, L. C. Purification of astrocytes from transgenic rodents by fluorescence-activated cell sorting. Cold Spring Harb.Protoc. 2013, 551-560 (2013).
- Smith, C., Otto, P., Bitner, R. A silica membrane-based method for the isolation of genomic DNA from tissues and cultured cells. CSH. Protoc. 2006, 2006-201 (2006).
- Sambrook, J., Russell, D. W. A Single-step Method for the Simultaneous Preparation. of DNA, RNA, and Protein from Cells and. 1, 2006-201 (2006).
- Barbas, C. F., Burton, D. R., Scott, J. K. . Quantitation of DNA and. , (2007).
- Zhao, Z., Han, L. CpG islands: algorithms and applications in methylation studies. Biochem. Biophys. Res. Commun. 382, 643-645 (2009).
- Srivastava, G. P., Guo, J., Shi, H. PRIMEGENS-v2: genome-wide primer design for analyzing DNA methylation patterns of CpG islands. Bioinformatics. 24, 1837-1842 (2008).
- Patterson, K., Molloy, L., Qu, W. DNA methylation: bisulphite modification and analysis. J.Vis.Exp.(56), doi:3170 [pii];10.3791/3170. , (2011).
- Drummond, G. B., Vowler, S. L. Categorized or continuous? Strength of an association-and linear regression. Adv.Physiol Educ. 36, 89-92 (2012).
- Sambrook, J., Russell, D. W. The basic polymerase chain reaction. CSH.Protoc. 1, (2006).
- Arpa, P. Strategies for cloning PCR products. Cold Spring Harb.Protoc. 8, (2009).
- Makovets, S. Basic DNA electrophoresis in molecular cloning: a comprehensive guide for beginners. Methods Mol.Biol. 1054, 11-43 (2013).
- Cahoy, J. D., et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neuroscience. 28, 264-278 (2008).
- Maldonado, P. P., Velez-Fort, M., Levavasseur, F. Oligodendrocyte precursor cells are accurate sensors of local K+ in mature gray matter. J Neuroscience. 33, 2432-2442 (2013).
- Kalsi, A. S., Greenwood, K., Wilkin, G. Kir4.1 expression by astrocytes and oligodendrocytes in CNS white matter: a developmental study in the rat optic nerve. J.Anat. 204, 475-485 (2004).
- Higashi, K., et al. An inwardly rectifying K(+) channel, Kir4.1, expressed in astrocytes surrounds synapses and blood vessels in brain. Am.J.Physiol Cell Physiol. 281 (3), (2001).
- Olsen, M. L., Higashimori, H., Campbell, S. L., Hablitz, J. J. Functional expression of Kir4.1 channels in spinal cord astrocytes. Glia. 53 (5), 516-528 (2006).
- Neusch, C., Rozengurt, N., Jacobs, R. E., Lester, H. A. Kir4.1 Potassium Channel Subunit Is Crucial for Oligodendrocyte Development and In Vivo Myelination. J. Neuroscience. 21 (15), 5429-5438 (2001).
- Kofuji, P., et al. Genetic Inactivation of an Inwardly Rectifying Potassium Channel (Kir4.1 Subunit) in Mice: Phenotypic Impact in Retina. J. Neuroscience. 20 (15), 5733-5740 (2000).
- Kofuji, P., Connors, N. C. Molecular substrates of potassium spatial buffering in glial cells. Mol.Neurobiol. 28, 195-208 (2003).
- Nwaobi, S. E., Lin, E., Peramsetty, S. R. DNA methylation functions as a critical regulator of Kir4.1 expression during CNS development. Glia. 62 (3), 411-427 (2014).
- Wojdacz, T. K., Dobrovic, A. Methylation-sensitive high-resolution melting. Nat.Protoc. 3, 1903-1908 (2008).
- Wojdacz, T. K., Dobrovic, A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 35, 10-1093 (2007).
- Laird, P. W. Principles and challenges of genomewide DNA methylation analysis. Nat.Rev.Genet. 11, 191-203 (2010).
