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{{Time_Course
{{TimeCourse
|collaborators=Boris Lenhard, Ana Pombo
|TCOverview='''Objective'''<br>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].<br><br>'''Background'''<br>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].<br><br>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.<br><br>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.<br><br>Significance<br>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.<br><br>References:<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>[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.<br>
|description=ES embryonic stem cell series
|TCQuality_control=<html><img src='/resource_browser/images/TC_qc/1000px-Mouse_visual_cortex.png' onclick='javascript:window.open("/resource_browser/images/TC_qc/1000px-Mouse_visual_cortex.png", "imgwindow", "width=1000,height=500");' style='width:700px;cursor:pointer'/></html><br>Figure 1: CAGE expression of marker genes in TPM.<br>* Pvalb - ref up regulated (parvalbumin neuron-specific marker)* Nav1 - down regulated (Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration. "expression is largely restricted to the NS during development." PMID: 15797708)
|libraryids=CNhs14085,CNhs14086,CNhs14087,CNhs14088,CNhs14089,CNhs14090,CNhs14091,CNhs14092,CNhs14093,CNhs14094,CNhs14095,CNhs14097,CNhs14098,CNhs14099,CNhs14100,CNhs14101,CNhs14102,CNhs14103,CNhs14104,CNhs14105,CNhs14106,CNhs14107,CNhs14108,CNhs14109,CNhs14110,CNhs14111,CNhs14112,CNhs14113,CNhs14114,CNhs14115,CNhs14116,CNhs14117,CNhs14118,CNhs14119,CNhs14121,CNhs14122,CNhs14123,CNhs14124,CNhs14125,CNhs14126,CNhs14127
|TCSample_description=RNA was extracted from the visual cortex dissected from wildtype and Mecp2 KO mice (B6;129P2-Mecp2tm1Bird/J) at the ages P14, P30 and P60. Dissected tissue was flash frozen in liquid nitrogen and shipped on Dry ice. RNA was extracted using Trizol and Qiagen RNAeasy columns with some modifications designed to retain small RNAs. Table 1 depicts the numbers of samples used.<br><table class='wikitable'><tr><th>Sample type</th><th>wild type</th><th>MeCP2 knockout (bird Model) </th></tr><tr><td>Visual cortex P14</td><td>4</td><td>3</tr><tr><td>Visual cortex P30</td><td>3</td><td>3</tr><tr><td>Visual cortex P60</td><td>3</td><td>3</tr><tr><td>Total samples</td><td>10</td><td>9</td></tr></table><br>Table-1: Number of samples in each subgroup in the visual cortex time course<br>
|Time_Course=
|category_treatment=Development
|collaborators=Michela Fagiolini
|description=mouse_visual_cortex
|germ_layer=ectoderm
|libraryids=CNhs13031,CNhs13032,CNhs13033,CNhs13034,CNhs13035,CNhs13036,CNhs13037,CNhs13038,CNhs13039,CNhs13040,CNhs13041,CNhs13042,CNhs13043,CNhs13044,CNhs13045,CNhs13046,CNhs13048,CNhs13820,CNhs13821
|number_time_points=3
|page_name=mouse_visualcortex
|page_name=mouse_visualcortex
|series=IN_VITRO DIFFERENTIATION SERIES
|primary_cells=primary cells
|series=DEVELOPMENTAL TISSUE SERIES
|species=Mouse (Mus musculus)
|species=Mouse (Mus musculus)
|zenbu_config=https://fantom5-collaboration.gsc.riken.jp/zenbu/gLyphs/#config
|tet_config=https://fantom.gsc.riken.jp/5/suppl/tet/Visualcortex.tsv.gz
|tet_file=https://fantom.gsc.riken.jp/5/tet#!/search/?filename=mm9.cage_peak_phase1and2combined_tpm_ann_decoded.osc.txt.gz&file=1&c=1&c=1018&c=1019&c=1020&c=1021&c=1022&c=1023&c=1024&c=1025&c=1026&c=1027&c=1028&c=1029&c=1030&c=1031&c=1032&c=1033&c=1034&c=1035&c=1036
|time_points=
|time_span=55 days
|timepoint_design=Embryonic stages
|tissue_cell_type=Visual cortex
|zenbu_config=https://fantom.gsc.riken.jp/zenbu/gLyphs/#config=U35NQvFpKNEnQqoNifwHl
}}
}}

Latest revision as of 17:44, 14 March 2022

Series:DEVELOPMENTAL TISSUE SERIES
Species:Mouse (Mus musculus)
Genomic View:Zenbu
Expression table:FILE
Link to TET:TET
Sample providers :Michela Fagiolini
Germ layer:ectoderm
Primary cells or cell line:primary cells
Time span:55 days
Number of time points:3


Overview

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.

Sample description
RNA was extracted from the visual cortex dissected from wildtype and Mecp2 KO mice (B6;129P2-Mecp2tm1Bird/J) at the ages P14, P30 and P60. Dissected tissue was flash frozen in liquid nitrogen and shipped on Dry ice. RNA was extracted using Trizol and Qiagen RNAeasy columns with some modifications designed to retain small RNAs. Table 1 depicts the numbers of samples used.
Sample typewild typeMeCP2 knockout (bird Model)
Visual cortex P1443
Visual cortex P3033
Visual cortex P6033
Total samples109

Table-1: Number of samples in each subgroup in the visual cortex time course
Quality control


Figure 1: CAGE expression of marker genes in TPM.
* Pvalb - ref up regulated (parvalbumin neuron-specific marker)* Nav1 - down regulated (Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration. "expression is largely restricted to the NS during development." PMID: 15797708)

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples



10235-104A1visual cortex - wildtypeneonate N15donor1
10236-104A2visual cortex - wildtypeneonate N15donor2
10237-104A3visual cortex - wildtypeneonate N15donor3
10238-104A4visual cortex - wildtypeneonate N30donor1
10239-104A5visual cortex - wildtypeneonate N30donor2
10240-104A6visual cortex - wildtypeneonate N30donor3
10241-104A7visual cortex - wildtypeneonate N60-70donor1
10242-104A8visual cortex - wildtypeneonate N60-70donor2
10243-104A9visual cortex - wildtypeneonate N60-70donor3
10244-104B1visual cortex - Mecp knockoutneonate N15donor1
10245-104B2visual cortex - Mecp knockoutneonate N15donor2
10246-104B3visual cortex - Mecp knockoutneonate N30donor1
10247-104B4visual cortex - Mecp knockoutneonate N30donor2
10248-104B5visual cortex - Mecp knockoutneonate N30donor3
10249-104B6visual cortex - Mecp knockoutneonate N60-70donor1
10250-104B7visual cortex - Mecp knockoutneonate N60-70donor2
10251-104B8visual cortex - Mecp knockoutneonate N60-70donor3
10349-105D7visual cortex - wildtypeneonate N15donor5
10350-105D8visual cortex - Mecp knockoutneonate N15donor3