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Objective
The main objective of this project was to identify differentially expressed genes in the visual cortex of the Mecp2 knockout (KO) mouse at three developmental ages ( Postnatal day 14 (P14), P30 and P60) critical for the maturation and regression of visual system and to characterize any variations in TSS usage in the Mecp2 KO mouse during development using the newly developed Helicos-CAGE technology [1].
Background
Mutations in the Methyl CpG binding protein 2 (MECP2) gene cause Rett Syndrome, a severe neuro-developmental disorder caused by impaired synaptic plasticity [2]. MeCP2, a member of the Methyl CpG binding domain family of proteins, is able to bind methylated and non-methylated DNA [3-6]. The MeCP2-DNA interaction results in chromatin compaction, which is correlated with silencing of chromatin. Binding of MeCP2 to chromatin also stabilizes the chromatin structure and protects from nuclease digestion [3].
The role of MeCP2 in transcription regulation has been studied extensively. ChIP-Chip studies done on differentiated SHSY5Y cells on customized microarrays reveal that MeCP2 binds mainly to inter-genic regions and that most promoters associated with MeCP2 are transcriptionally active [7]. Studies done on the expression profile of Mecp2 null, Mecp2 transgenic (tg) and wild type animals reveal that 85% of the total genes mis-regulated in mecp2 mouse models show a profile consistent with an unexpected transcriptional activator-like role for Mecp2 [8]. These data obtained from mouse hypothalamus, were confirmed by extensive quantitative PCR analysis and Mecp2 binding was confirmed with ChIP-qPCR. Bisulfite sequencing analysis showed that active MeCP2 target promoters were not methylated [8]. However, recent ChIP-seq studies conducted in brain revealed that Mecp2 binds all over the genome in a uniform manner but shows specific enrichment peaks in methylated regions [9, 10] while ChIP-seq in astrocytes reveals that MeCP2 binds to specific gene targets [11] thus suggesting a cell specific role for MeCP2. Altogether there is uncertainty about the role of MeCP2 in transcriptional regulation and binding to specific targets. MeCP2 appears to bind transcriptionally active as well as inactive regions of the genome; but whether it participates in the regulation of expression of specific target genes needs more investigation. If MeCP2 regulates the transcription of key genes involved in synaptic plasticity, it is important to identify them for the design of therapeutic targeting strategies and to gain insights into the molecular pathogenesis of Rett Syndrome and other disorders associated with MECP2 mutations. Our project aimed to address this uncertainty by using state of the art sequencers and newly developed “omics” technology.
The mouse visual cortex provides an extraordinary opportunity in this research. Plasticity in the visual cortex can be manipulated by dark /light rearing of mice and studied using standard electrophysiological techniques and this tissue holds immense promise as a system for manipulation and monitoring of plasticity through therapeutic drugs and targeted gene therapy. Physiological and behavioral studies conducted by Dr. Michela Fagiolini on the visual cortex of mecp2 null male mice reveal an "apparent" normal development of visual cortical plasticity from P14 until P30 when vision rapidly begins to regress together with the onset of the general Rett syndrome phenotype. By P60 Mecp2 KO mice exhibit a full regression [12]. These three ages also correspond to three phases of visual cortex development and plasticity: pre-critical period, critical period and adulthood. Thus the visual cortex represents a characterized region of the brain in terms of plasticity impairment related to Mecp2 deficiency and we aimed to conduct our studies in the visual cortex of male wild type and mecp2 null mice. In this project we analyzed the transcriptome at the three critical stages of visual cortex development: P14, P30 and P60; which is expected to provide us with a comprehensive picture of the transcriptional irregularities that commence before and after the onset of electrophysiological changes.
Significance
Our studies have the potential to identify genes mis-regulated before the onset of behavioral changes, track changes in gene expression as development proceeds and also identify any variations in transcriptional start site usage before and after the onset of symptoms in the mouse visual cortex. At its conclusion, it will provide a comprehensive understanding of the molecular pathways leading to impairment in developmental plasticity in the visual cortex of mecp2 null mice. We expect to identify a few key genes / pathways involved in synaptic plasticity and regulated by mecp2 which will provide future directions towards targeted therapy for this disorder.
References:
[1] Kanamori-Katayama M, Itoh M, Kawaji H, Lassmann T, Katayama S, Kojima M, Bertin N, Kaiho A, Ninomiya N, Daub CO et al: Unamplified cap analysis of gene expression on a single-molecule sequencer. Genome Res 2011, 21(7):1150-1159.
[2] Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY: Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 1999, 23(2):185-188.
[3] Georgel PT, Horowitz-Scherer RA, Adkins N, Woodcock CL, Wade PA, Hansen JC: Chromatin compaction by human MeCP2. Assembly of novel secondary chromatin structures in the absence of DNA methylation. J Biol Chem 2003, 278(34):32181-32188.
[4] Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe AP: Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 1998, 19(2):187-191.
[5] Nan X, Tate P, Li E, Bird A: DNA methylation specifies chromosomal localization of MeCP2. Mol Cell Biol 1996, 16(1):414-421.[6] Nikitina T, Shi X, Ghosh RP, Horowitz-Scherer RA, Hansen JC, Woodcock CL: Multiple modes of interaction between the methylated DNA binding protein MeCP2 and chromatin. Mol Cell Biol 2007, 27(3):864-877.
[7] Yasui DH, Peddada S, Bieda MC, Vallero RO, Hogart A, Nagarajan RP, Thatcher KN, Farnham PJ, Lasalle JM: Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes. Proc Natl Acad Sci U S A 2007, 104(49):19416-19421.
[8] Chahrour M, Jung SY, Shaw C, Zhou X, Wong ST, Qin J, Zoghbi HY: MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 2008, 320(5880):1224-1229.
[9] Cohen S, Gabel HW, Hemberg M, Hutchinson AN, Sadacca LA, Ebert DH, Harmin DA, Greenberg RS, Verdine VK, Zhou Z et al: Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function. Neuron 2011, 72(1):72-85.
[10] Skene PJ, Illingworth RS, Webb S, Kerr AR, James KD, Turner DJ, Andrews R, Bird AP: Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state. Mol Cell 2010, 37(4):457-468.
[11] Yasui DH, Xu H, Dunaway KW, Lasalle JM, Jin LW, Maezawa I: MeCP2 modulates gene expression pathways in astrocytes. Mol Autism 2013, 4(1):3.
[12] Durand S, Patrizi A, Quast KB, Hachigian L, Pavlyuk R, Saxena A, Carninci P, Hensch TK, Fagiolini M: NMDA Receptor Regulation Prevents Regression of Visual Cortical Function in the Absence of Mecp2. Neuron 2012, 76(6):1078-1090.
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