Limits...
Over-expression of either MECP2_e1 or MECP2_e2 in neuronally differentiated cells results in different patterns of gene expression.

Orlic-Milacic M, Kaufman L, Mikhailov A, Cheung AY, Mahmood H, Ellis J, Gianakopoulos PJ, Minassian BA, Vincent JB - PLoS ONE (2014)

Bottom Line: We used quantitative RT-PCR to verify microarray results for 41 of these genes.We found significant up-regulation of several genes resulting from over-expression of MECP2_e1 including SRPX2, NAV3, NPY1R, SYN3, and SEMA3D.Understanding the biology of these differentially transcribed genes and their role in neurodevelopment may help us to understand the relative functions of the two MECP2 isoforms, and ultimately develop a better understanding of RTT etiology and determine the clinical relevance of isoform-specific mutations.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropsychiatry & Development Lab, Campbell Family Mental Health Research Institute, The Centre for Addiction & Mental Health, Toronto, Ontario, Canada.

ABSTRACT
Mutations in MECP2 are responsible for the majority of Rett syndrome cases. MECP2 is a regulator of transcription, and has two isoforms, MECP2_e1 and MECP2_e2. There is accumulating evidence that MECP2_e1 is the etiologically relevant variant for Rett. In this study we aim to detect genes that are differentially transcribed in neuronal cells over-expressing either of these two MECP2 isoforms. The human neuroblastoma cell line SK-N-SH was stably infected by lentiviral vectors over-expressing MECP2_e1, MECP2_e2, or eGFP, and were then differentiated into neurons. The same lentiviral constructs were also used to infect mouse Mecp2 knockout (Mecp2(tm1.1Bird)) fibroblasts. RNA from these cells was used for microarray gene expression analysis. For the human neuronal cells, ∼ 800 genes showed >three-fold change in expression level with the MECP2_e1 construct, and ∼ 230 with MECP2_e2 (unpaired t-test, uncorrected p value <0.05). We used quantitative RT-PCR to verify microarray results for 41 of these genes. We found significant up-regulation of several genes resulting from over-expression of MECP2_e1 including SRPX2, NAV3, NPY1R, SYN3, and SEMA3D. DOCK8 was shown via microarray and qRT-PCR to be upregulated in both SK-N-SH cells and mouse fibroblasts. Both isoforms up-regulated GABRA2, KCNA1, FOXG1 and FOXP2. Down-regulation of expression in the presence of MECP2_e1 was seen with UNC5C and RPH3A. Understanding the biology of these differentially transcribed genes and their role in neurodevelopment may help us to understand the relative functions of the two MECP2 isoforms, and ultimately develop a better understanding of RTT etiology and determine the clinical relevance of isoform-specific mutations.

Show MeSH

Related in: MedlinePlus

In SK-N-SH cells, MECP2_e1 induces expression of A) FOXP2 and B) DOCK8.A weak signal is also visible when cells are infected with MECP2_e2 lentivirus vector. Frontal cortex was used as a positive control. Replicates represent biological replicates i.e. repeated infections. For FOXP2, real-time RT-PCR products were run on an agarose gel. For DOCK8, end point PCR was performed using KAPA2G Fast HotStartReadyMix (KapaBiosystems, KK5601. PCR consisted of 35 cycles, and the annealing temperature was 60°C. C) GAPDH was used as an endogenous control for RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3974668&req=5

pone-0091742-g005: In SK-N-SH cells, MECP2_e1 induces expression of A) FOXP2 and B) DOCK8.A weak signal is also visible when cells are infected with MECP2_e2 lentivirus vector. Frontal cortex was used as a positive control. Replicates represent biological replicates i.e. repeated infections. For FOXP2, real-time RT-PCR products were run on an agarose gel. For DOCK8, end point PCR was performed using KAPA2G Fast HotStartReadyMix (KapaBiosystems, KK5601. PCR consisted of 35 cycles, and the annealing temperature was 60°C. C) GAPDH was used as an endogenous control for RT-PCR.

Mentions: We used quantitative RT-PCR to verify microarray results for a total of 41 genes, including 29 that, in addition to individually significant change in expression level (uncorrected for FDR), are known to be involved in cognitive function or neuronal development, or are highly expressed in CNS tissues, or are implicated in Rett syndrome, intellectual disability or autism (Tables 2, 3 and 4, Figures 4, and 5). An additional 12 genes were identified as differently expressed in both the transfected SK-N-SH cells and in the mouse knockout fibroblasts (Table 5). As uncorrected statistical tests were used to identify differentially expressed genes by microarray, a higher rate of false-positives and false-negatives was expected. We confirmed significant up-regulation of several genes resulting from over-expression of the e1 isoform in SK-N-SH cells, including NAV3 (5-fold level increase), FOXP2 (induced by MECP2_e1 over-expression, and weakly induced by MECP2_e2 (“induced”-indicating that no expression was detectable in control eGFP-infected cells, thus no fold-change calculation was possible), Figure 5a), NPY1R (six-fold increase), SYN3 (two-fold increase), and SEMA3D (six-fold increase). Both MECP2_e1 and MECP2_e2 up-regulated GABRA2 (18-fold and three-fold increase, respectively), KCNA1 (seven-fold and three-fold increase, respectively) and FOXG1 (six-fold and three-fold increase, respectively). Down-regulation of expression in the presence of e1 was seen with UNC5C(40% down-regulation) and RPH3A (30% down-regulation). Expression levels of four genes determined by the qPCR contradicted microarray results: SPRX2, ITGA3 and NDN were reported as down-regulated by microarray analysis but found to be up-regulated by qPCR, and CNTN4, which was identified as up-regulated by microarray analysis, was indicated as down-regulated by qPCR (Table 2). Several of the genes, while potentially relevant to RTT, had very low levels of expression and could not be validated by this method, including DLGAP2, CRH, GABRB, GRIN2A and PTCHD1 (Table 2).


Over-expression of either MECP2_e1 or MECP2_e2 in neuronally differentiated cells results in different patterns of gene expression.

Orlic-Milacic M, Kaufman L, Mikhailov A, Cheung AY, Mahmood H, Ellis J, Gianakopoulos PJ, Minassian BA, Vincent JB - PLoS ONE (2014)

In SK-N-SH cells, MECP2_e1 induces expression of A) FOXP2 and B) DOCK8.A weak signal is also visible when cells are infected with MECP2_e2 lentivirus vector. Frontal cortex was used as a positive control. Replicates represent biological replicates i.e. repeated infections. For FOXP2, real-time RT-PCR products were run on an agarose gel. For DOCK8, end point PCR was performed using KAPA2G Fast HotStartReadyMix (KapaBiosystems, KK5601. PCR consisted of 35 cycles, and the annealing temperature was 60°C. C) GAPDH was used as an endogenous control for RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974668&req=5

pone-0091742-g005: In SK-N-SH cells, MECP2_e1 induces expression of A) FOXP2 and B) DOCK8.A weak signal is also visible when cells are infected with MECP2_e2 lentivirus vector. Frontal cortex was used as a positive control. Replicates represent biological replicates i.e. repeated infections. For FOXP2, real-time RT-PCR products were run on an agarose gel. For DOCK8, end point PCR was performed using KAPA2G Fast HotStartReadyMix (KapaBiosystems, KK5601. PCR consisted of 35 cycles, and the annealing temperature was 60°C. C) GAPDH was used as an endogenous control for RT-PCR.
Mentions: We used quantitative RT-PCR to verify microarray results for a total of 41 genes, including 29 that, in addition to individually significant change in expression level (uncorrected for FDR), are known to be involved in cognitive function or neuronal development, or are highly expressed in CNS tissues, or are implicated in Rett syndrome, intellectual disability or autism (Tables 2, 3 and 4, Figures 4, and 5). An additional 12 genes were identified as differently expressed in both the transfected SK-N-SH cells and in the mouse knockout fibroblasts (Table 5). As uncorrected statistical tests were used to identify differentially expressed genes by microarray, a higher rate of false-positives and false-negatives was expected. We confirmed significant up-regulation of several genes resulting from over-expression of the e1 isoform in SK-N-SH cells, including NAV3 (5-fold level increase), FOXP2 (induced by MECP2_e1 over-expression, and weakly induced by MECP2_e2 (“induced”-indicating that no expression was detectable in control eGFP-infected cells, thus no fold-change calculation was possible), Figure 5a), NPY1R (six-fold increase), SYN3 (two-fold increase), and SEMA3D (six-fold increase). Both MECP2_e1 and MECP2_e2 up-regulated GABRA2 (18-fold and three-fold increase, respectively), KCNA1 (seven-fold and three-fold increase, respectively) and FOXG1 (six-fold and three-fold increase, respectively). Down-regulation of expression in the presence of e1 was seen with UNC5C(40% down-regulation) and RPH3A (30% down-regulation). Expression levels of four genes determined by the qPCR contradicted microarray results: SPRX2, ITGA3 and NDN were reported as down-regulated by microarray analysis but found to be up-regulated by qPCR, and CNTN4, which was identified as up-regulated by microarray analysis, was indicated as down-regulated by qPCR (Table 2). Several of the genes, while potentially relevant to RTT, had very low levels of expression and could not be validated by this method, including DLGAP2, CRH, GABRB, GRIN2A and PTCHD1 (Table 2).

Bottom Line: We used quantitative RT-PCR to verify microarray results for 41 of these genes.We found significant up-regulation of several genes resulting from over-expression of MECP2_e1 including SRPX2, NAV3, NPY1R, SYN3, and SEMA3D.Understanding the biology of these differentially transcribed genes and their role in neurodevelopment may help us to understand the relative functions of the two MECP2 isoforms, and ultimately develop a better understanding of RTT etiology and determine the clinical relevance of isoform-specific mutations.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropsychiatry & Development Lab, Campbell Family Mental Health Research Institute, The Centre for Addiction & Mental Health, Toronto, Ontario, Canada.

ABSTRACT
Mutations in MECP2 are responsible for the majority of Rett syndrome cases. MECP2 is a regulator of transcription, and has two isoforms, MECP2_e1 and MECP2_e2. There is accumulating evidence that MECP2_e1 is the etiologically relevant variant for Rett. In this study we aim to detect genes that are differentially transcribed in neuronal cells over-expressing either of these two MECP2 isoforms. The human neuroblastoma cell line SK-N-SH was stably infected by lentiviral vectors over-expressing MECP2_e1, MECP2_e2, or eGFP, and were then differentiated into neurons. The same lentiviral constructs were also used to infect mouse Mecp2 knockout (Mecp2(tm1.1Bird)) fibroblasts. RNA from these cells was used for microarray gene expression analysis. For the human neuronal cells, ∼ 800 genes showed >three-fold change in expression level with the MECP2_e1 construct, and ∼ 230 with MECP2_e2 (unpaired t-test, uncorrected p value <0.05). We used quantitative RT-PCR to verify microarray results for 41 of these genes. We found significant up-regulation of several genes resulting from over-expression of MECP2_e1 including SRPX2, NAV3, NPY1R, SYN3, and SEMA3D. DOCK8 was shown via microarray and qRT-PCR to be upregulated in both SK-N-SH cells and mouse fibroblasts. Both isoforms up-regulated GABRA2, KCNA1, FOXG1 and FOXP2. Down-regulation of expression in the presence of MECP2_e1 was seen with UNC5C and RPH3A. Understanding the biology of these differentially transcribed genes and their role in neurodevelopment may help us to understand the relative functions of the two MECP2 isoforms, and ultimately develop a better understanding of RTT etiology and determine the clinical relevance of isoform-specific mutations.

Show MeSH
Related in: MedlinePlus