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Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients
© Delgado et al; licensee BioMed Central Ltd. 2006
Received: 25 October 2005
Accepted: 21 July 2006
Published: 21 July 2006
More than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the wild type or mutant MECP2 in each of their cells. To test the hypothesis that MECP2 mutations result in genome-wide transcriptional deregulation and identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we performed gene expression profiling of pure populations of untransformed T-lymphocytes that express either a mutant or a wild-type allele.
Single T lymphocytes from a patient with a c.473C>T (p.T158M) mutation and one with a c.1308-1309delTC mutation were subcloned and subjected to short term culture. Gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis.
Expression profiling yielded 44 upregulated genes and 77 downregulated genes. We compared this gene list with expression profiles of independent microarray experiments in cells and tissues of RTT patients and mouse models with Mecp2 mutations. These comparisons identified a candidate MeCP2 target gene, SPOCK1, downregulated in two independent microarray experiments, but its expression was not altered by quantitative RT-PCR analysis on brain tissues from a RTT mouse model.
Initial expression profiling from T-cell clones of RTT patients identified a list of potential MeCP2 target genes. Further detailed analysis and comparison to independent microarray experiments did not confirm significantly altered expression of most candidate genes. These results are consistent with other reported data.
Rett syndrome (RTT, OMIM 312750) is an X-linked neurodevelopmental disorder that affects 1 in 10,000 to 15,000 females [1, 2]. Girls with RTT have an apparently normal early development, followed by deceleration of head growth, loss of language skills, loss of purposeful hand movements and impaired social contact. As the disease progresses they develop respiratory abnormalities, autistic features, stereotypic hand movements, scoliosis, general growth delay, seizures and ataxia [3, 4]. RTT is caused by heterozygous mutations in the methyl-CpG-binding protein 2 gene (MECP2), an X-linked gene subject to X chromosome inactivation (XCI) . Mutations in the coding region of this gene are detected in 85% of patients with classic RTT [6–9]. An additional 10% have large deletions affecting several exons of MECP2 [10–12]. Alternative splice variants of MECP2 have been identified [9, 13, 14] that result in two protein isoforms. MeCP2-e1 (MeCP2α/B) is encoded by exons 1, 3 and 4 and is more abundant in brain than the previously identified MeCP2-e2 (MeCP2β/A) isoform, which is encoded by exons 2, 3 and 4. Interestingly, mutations in exon 1 are only rarely found in RTT patients [9, 15, 16]. Both isoforms of MeCP2 are identical beyond exon 2 and contain an 84-amino acid methyl-CpG-binding domain  and a 104-amino acid transcriptional repression domain (TRD)  as well as a C-terminal protein interaction domain. MeCP2 has been shown to bind DNA, preferentially at methylated CpG dinucleotides with resulting transcriptional repression of nearby genes through the recruitment of a histone deacetylase (HDAC1 and 2) and a Sin3A-containing corepressor complex [19, 20]. MeCP2 also associates with histone methyltransferase activity and the DNA methyltransferase DNMT1 [21, 22]. Brahma (Brm), the catalytic component of the SWI/SNF ATPase-dependent remodelling complex, was found to interact with MeCP2 , extending the mechanistic link between DNA methylation, chromatin remodelling and transcriptional repression. Recently, MecP2 has also been demonstrated to regulate alternative splicing and interact with an RNA-binding protein (Y box-binding protein 1) .
Despite active research since the discovery of MECP2 mutations in RTT, it has proven difficult to identify other direct target genes for the proposed functions of MeCP2. Candidate gene-based approaches using vertebrate models with disrupted MeCP2 have resulted in the identification of brain-derived neurotrophic factor (Bdnf) [25–27] and Hairy2a  as MeCP2 targets. MeCP2 binds to methylated CpG sites near promoter III of BDNF in resting neurons [25, 26], and disease progression in a RTT mouse model correlates inversely with Bdnf expression . Hairy2a is upregulated in the absence of MeCP2 in Xenopus embryos . Following the hypothesis that MeCP2 functions primarily as a transcriptional repressor, several groups have attempted to screen for its targets by transcriptional profiling using RNA from postmortem brain tissues or cell lines derived from RTT patients, or from tissues of mice with engineered mutations in Mecp2. In one study, 70 transcripts were found to have altered gene expression in mutant versus wild-type fibroblast clones and lymphoblastoid cells lines . The authors concluded that MeCP2 deficiency did not lead to global deregulation of gene expression and suggested that clonal fibroblast lines may show substantial variation, making them an unstable resource for expression profiling studies. In addition, lymphoblastoid cell lines are immortalized by Epstein-Barr virus (EBV) transformation, which can alter their transcriptional profile and methylation status. Expression profiling of brain from male mice with a deletion of Mecp2 also yielded only few genes with altered expression between wild-type and mutant mice . Upon further analysis, those identified fell well within the range of the high false-positive rate . In a third transcriptional profiling microarray study on postmortem RTT brains, significant changes in expression for 135 genes on three different cDNA microarrays were found . Yet, the small sample size analyzed and the validation of the array data with the same samples instead of independent samples may have led to false-positive results . It has been hypothesized that transcriptional profiling of MECP2-deficient brains has failed to identify MeCP2-regulated genes because of the high complexity in regional organization and the admixture of neuronal and non-neuronal cell types that is inherent to brain tissue . Perhaps MeCP2 targets are also difficult to identify by microarray analysis because the absolute changes in expression level for individual genes are very small . A more direct approach that circumvents this problem is the use of a chromatin immunoprecipitation (ChIP) strategy to directly search for the sites of MeCP2-binding to DNA. This led to the recent identification of the DLX5 gene as a MeCP2 target and other genes such as CNTN2, FOXA3, and SIAT4A as candidate genes regulated by MeCP2 .
In the present study we sought to overcome some of the limitations of transcriptional profiling on complex tissues, such as brain, as well as those of transformed lymphoblastoid cell lines by studying clonal cultures of non-transformed lymphocytes from individual RTT patients. These cells can be easily obtained through blood sampling, readily cultured and subjected to single-cell subcloning. Because MECP2 undergoes XCI, this last feature allows the separation of cells that express the wild-type MECP2 from the active X from those cells that express the mutant MECP2 from the active X. To identify downstream targets of MeCP2, we compared global gene expression patterns in matched pairs of clonally derived mutant or wild-type MECP2-expressing lymphocyte cultures from two girls with classic Rett syndrome. Our microarray analysis revealed 121 genes with changes in expression between cells expressing the wild-type MECP2 and those expressing the mutant MECP2. We compared this gene list to those of other transcriptional profiling experiments and focused further analysis on a downregulated putative MeCP2 target, SPOCK1. However, quantitative RT-PCR analysis on RNA from the clonal T-lymphocytes, differentiating mouse embryoid bodies (EB) and various mouse brain regions did not reveal significantly different expression between mutant and wild-type RNAs.
T-lymphocyte single cell cloning
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque from fresh blood samples of Rett syndrome patients, collected under a research protocol approved by the Baylor College of Medicine Institutional Review Board for Human Subjects Research. Isolation of T-lymphocyte clones has been described previously [35–37]. Briefly, PBMCs from the patients were plated at limiting dilution in 96-well plates in the presence of irradiated allogeneic PBMCs in RPMI media containing 20% pooled human sera (Sigma-Aldrich, St. Louis, MO), 2 μg/mL PHA.P (Murex diagnostics, Inc. Dartford Kent, UK) and 5% Human T-stim (Collaborative Biomedical Products, Bedford, MA). Nine to ten days following original culture, wells showing clonal positive growth were restimulated and cultured for an additional 7–14 days. Additional restimulations were performed every 10–12 days until >107 cells were obtained from each clone.
RNA isolation and reverse transcription
Total RNA was isolated from T-cell clones using Trizol reagent (Invitrogen Corp., Carlsbad, CA). RNA was treated with 1 U of RNase-free DNase I (Ambion Inc., Austin, TX). RNA samples were stored at -80°C until later use. Analysis of allelic expression of MECP2 by RT-PCR and restriction digestion to verify clonality of expanded lymphocyte clones from patients RT208 (c.1308-1309delTC mutation) and RT211 (p.T158M mutation) has been previously described .
Animal research was performed under a protocol approved by the Baylor College of Medicine animal research review board. After euthanasia, brain regions (cortex, cerebellum, olfactory bulb) were quickly removed from 5 week-old mice and immediately frozen in liquid nitrogen. E16.5 embryos were collected and heads and bodies immediately frozen in liquid nitrogen. Total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen Inc., Valencia, CA). Prior to array hybridization, the RNA quality and degradation was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). In addition, quality control parameters were assessed throughout the experimental process to assure the efficiency of transcription, integrity of hybridization, and consistency of qualitative calls. Assessment during the synthesis of the hybridization transcript was accomplished by spectrophotometric analysis of the starting RNA and cRNA, and by gel electrophoresis following synthesis of the cDNA, cRNA and fragmentation of the cRNA. A 3'/5' ratio of GAPDH less than 2 was considered acceptable for efficiency of transcription. Spiked control transcripts were also monitored to verify hybridization integrity. Scaling factors for each sample ranged between 1.72 and 5.39. All quality control measures were consistent with the manufacturers recommended procedures and conformed to the recommended cut-off values except for one of the RT211 (p.T158M) wild-type clonal lymphocyte samples (Wt5) which was not included in subsequent analysis because the RNA was found to be partially degraded.
Oligonucleotide expression array hybridization
Total RNA was converted to double-stranded cDNA (ds-cDNA) in the presence of an oligo-dT primer containing a T7 RNA polymerase promoter. In vitro transcription was performed to generate biotin-labeled cRNA from the ds-cDNA template in the presence of a mixture of biotin-labeled ribonucleotides. Biotin-labeled cRNA (15 μg) was fragmented to a size range between 50–200 bases in length and used to hybridize to Affymetrix human HG-U133A chips. After hybridization, the arrays were washed and stained with Streptavidin-phycoerythrin. Arrays were read at a resolution of 3 microns using a HP Gene Array Scanner (Hewlett Packard, Inc., Palo Alto CA) The average background for each chip was determined to be below the common threshold of 100 for all analyzed chips.
Microarray data analysis
Gene lists of all up and downregulated genes at the p < 0.05 level were then compared to lists of genes from analysis of another transcriptional profiling experiment in progress in our laboratory that compares gene expression profiles of Mecp2 R308/Y -mutant  and wild-type embryoid bodies (EB) at day 5 after retinoic acid-induced in vitro differentiation of mouse embryonic stem cells (unpublished data). We also compared the gene lists from the clonal lymphocyte cultures to those from previously published microarray experiments on RNA samples of Rett syndrome patients or of Mecp2-mutant mouse models [29–31, 44], and genes found to be MeCP2-targets by chromatin immunoprecipitation experiments . To allow direct comparison of all different datasets using consistent nomenclature, we replaced the gene annotations in all these lists with the official symbol for the human genes (or human orthologue of mouse genes) from the NCBI Entrez Gene browser  (See additional file 1: supplementary table 1).
Quantitative real-time PCR
For quantitative real-time PCR on mouse tissues, an equal amount of total RNA isolated from brain regions (cortex, cerebellum and olfactory bulb) of 5 week-old mice or E16.5 embryos was converted to cDNA with SuperScript II reverse transcriptase (Invitrogen Corp., Carlsbad, CA). For quantitative real-time PCR on human samples, total RNA from the clonal lymphocyte cultures used for microarray hybridization was similarly converted to cDNA. We used the ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA) to perform quantitative real time PCR with SYBR Green as the detection agent. The primers used were as follows. Murine Spock1 (NM_009262): Spock1 forward primer: TGCACGGACAAGGAGCTGCG, Spock1 reverse primer: GAACCAGTCCTTCAGCCGG; Murine Gapdh (NM_001001303): Gapdh forward primer: CATGGCCTTCCGTGTTCCTA, Gapdh reverse primer: GCGGCACGTCAGATCCA; human SPOCK1 (NM_004598): SPOCK1 forward primer: TGCACAGACAAGGAGTTGCG, SPOCK1 reverse primer: AAACCAATCCTTCAGCCGG; human GAPDH (NM_002046): GAPDH forward primer: TGGGCTACACTGAGCACCAG, and GAPDH reverse primer: GGGTGTCGCTGTTGAAGTCA. After PCR amplification, a dissociation protocol was performed to determine the melting curve of the PCR product. Only reactions with melting curves indicative of a single amplification product were analyzed further. The identity and expected size of the single PCR product was also confirmed by agarose gel electrophoresis. Relative quantification of the abundance of each gene at every time point was performed by the comparative ΔΔCT method as described in the Applied Biosystem user bulletin #2 . The values obtained by this method are a measure of the fold-change in expression of the gene of interest compared to the calibrator sample, all normalized to Gapdh (mouse samples) or GAPDH (human samples). The Student's t test was used to determine statistical significance of expression differences between average CT values of wild-type and mutant samples. A p-value < 0.05 was considered significant.
Isolation of MECP2-mutant and MECP2-wild type T-lymphocyte clones
Because MECP2 is an X-linked gene that undergoes XCI in females, each cell expresses MECP2 exclusively from only one of the two X chromosomes (the active X), while the copy of the gene on the inactive X chromosome is silenced. In order to separate cells that express the wild type MECP2 from those that express the mutant MECP2 allele, we performed single-cell cloning of T-lymphocytes isolated from peripheral blood mononuclear cells of RTT patients . To allow for comparison between two types of mutations, samples from two girls with classic RTT, one (RT211) with a p.T158M missense mutation and one (RT208) with a c.1308-1309delTC frameshift mutation, were used for this experiment. Following two rounds of mitogenic stimulation, all clonal cultures with greater than 107 cells were harvested for RNA isolation. As previously reported for these cell cultures, RT-PCR followed by digestion of the amplified products to completion with restriction enzymes specific for the mutant allele, demonstrated that we had obtained pure populations of clonal cells, expressing either a mutant or a wild-type MECP2 .
Microarray analysis of matched lymphocyte clones
Two technical replicate hybridizations were performed for one of each clone type, as well as an additional biological replicate hybridization for the wild-type MECP2 and mutant MECP2 clones of RT211 (p.T158M) on HG-U133A human genome chips containing 22,283 genes (Affymetrix, Santa Clara, CA) (Figure 1). Signals of all hybridized chips were normalized using the GC-RMA method . We then performed one-way ANOVA analysis to find genes with significantly altered expression in Mut2 and Mut4 from patient RT208 (c.1308-1309delTC), and Mut1, Mut3 and Mut5 from patient RT211 (p.T158M) compared to WT2 and WT4 from patient RT208, and WT1 and WT3 from patient RT211.
Genes with lower expression in mutant samples on microarray analysis. This table contains a list of genes with lower expression in mutant T-lymphocyte clones by one-way ANOVA analysis (p < 0.05). * indicates >1.5-fold change in expression level and ** indicates >2-fold change in expression level.
cold shock domain protein A
SRY (sex determining region-Y) box 4
Transcribed seq with strong similarity BIR1
glutathione S-transferase M1
natural cytotoxicity triggering receptor 3
transmembrane 4 superfamily member 13
G protein-coupled receptor 15
hypothetical protein FLJ10097
T cell receptor beta chain BV20S1 BJ1-5 BC1 mRNA
FXYD domain containing ion transport regulator 7
glutathione S-transferase M1
non-metastatic cells 4, protein expressed in
protein tyrosine phosphatase, receptor type N polypeptide 2
1-acylglycerol-3-phosphate O-acyltransferase 4
protein tyrosine phosphatase-like, member a
RAB31, member RAS oncogene family
regulatory factor X, 3 (Influences HLA class II expression)
Fc fragment of IgE, high affinity receptor for γ-polypeptide
chemokine (C-C motif) ligand 5
adrenergic, beta-2-, receptor, surface
natural cytotoxicity triggering receptor 3
chromosome 14 open reading frame 2
type 1 TNF receptor shedding aminopeptidase regulator
G protein-coupled receptor kinase 5
natural cytotoxicity triggering receptor 3
chloride intracellular channel 3
hypothetical protein LOC283687
GTPase activating RANGAP domain-like 4
solute carrier family 16, member 1
CDNA FLJ13754 fis, clone PLACE3000362
HUS1 checkpoint homolog (S. pombe)
nerve growth factor receptor (TNFRSF16) ass. protein 1
G protein-coupled receptor 56
sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican)
chemokine (C-X-C motif) receptor 6
actin binding LIM protein 1
Ras and Rab interactor 3
natural killer cell group 7 sequence
AV705559ADB Homo sapiens cDNA clone ADBAPE04 5'
phosphatidylinositol transfer protein, cytoplasmic 1
Genes with higher expression in mutant samples on microarray analysis. This table contains a list of genes with higher expression in mutant T-lymphocyte clones by one-way ANOVA analysis (p < 0.05). * indicates >1.5-fold change in expression level and ** indicates >2-fold change in expression level.
leukocyte-derived arginine aminopeptidase
interleukin 3 receptor, alpha (low affinity)
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein
hypothetical protein FLJ10808
catenin (cadherin-associated protein), alpha 1, 102 kDa
lectin, galactoside-binding, soluble, 8 (galectin 8)
enoyl Coenzyme A hydratase domain containing 1
protein kinase (cAMP-dependent, catalytic) inhibitor γ
glutathione S-transferase M2 (muscle)
coronin, actin binding protein, 1B
hypothetical protein FLJ10116
ras homolog gene family, member B
T-cell lymphoma invasion and metastasis 1
catenin (cadherin-associated protein), alpha 1, 102 kDa
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein
protein tyrosine phosphatase, non-receptor type 13
transcription factor 4
phosphodiesterase 4D interacting protein (myomegalin)
myelin basic protein
triple functional domain (PTPRF interacting)
TBC1 domain family, member 2
DnaJ (Hsp40) homolog, subfamily D, member 1
Human mRNA for HuIFN-gamma interferon.
PERP, TP53 apoptosis effector
ubiquitin protein ligase E3B
neurofilament, heavy polypeptide 200 kDa
eukaryotic translation initiation factor 3, subunit 8, 110 kDa
homolog of C. elegans smu-1
COX15 homolog, cytochrome c oxidase assembly protein
NEL-like 2 (chicken)
anterior pharynx defective 1B-like
T-cell receptor precursor
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide
catenin, beta like 1
major histocompatibility complex, class II, DR beta 3
eukaryotic translation initiation factor 3, subunit 8, 110 kDa
hypothetical protein FLJ21616
chemokine (C-C motif) ligand 1
acyl-CoA synthetase long-chain family member 1
RNA binding motif protein 8A
eukaryotic translation initiation factor 3, subunit 8, 110 kDa
eukaryotic translation elongation factor 1 delta
Rab6-interacting protein 2
hypothetical protein MGC2654
caspase 6, apoptosis-related cysteine protease
eukaryotic translation initiation factor 2, subunit 1 alpha, 35 kDa
zinc finger protein 36, C3H type-like 1
ATPase, Na+/K+ transporting, beta 1 polypeptide
ym27c01.s1 Soares infant brain 1NIB cDNA clone IMAGE:49385 3'
ADP-ribosylation factor 6
AL541302 Homo sapiens PLACENTA cDNA clone CS0DE006YI10 5'
dual specificity phosphatase 4
chemokine (C motif) ligand 1
zinc finger, BED domain containing 2
minor histocompatibility antigen HB-1
phosphoribosyl pyrophosphate amidotransferase
TAF9-like RNA polymerase II
target of myb1 (chicken)
Similar to PI-3-kinase-related kinase SMG-1 isoform 1; (LOC390682)
autism susceptibility candidate 2
ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin)
transmembrane 7 superfamily member 1 (upregulated in kidney)
DnaJ (Hsp40) homolog, subfamily B, member 6
triple functional domain (PTPRF interacting)
clone HQ0477 PRO0477p
zinc finger protein 131 (clone pHZ-10)
butyrophilin, subfamily 3, member A1
Expression of the mouse orthologue of SPOCK1is not altered in a RTT mouse model
Heterozygous mutations in X-linked MECP2 are found in most girls with RTT. The MECP2 gene is subject to XCI and classic RTT patients typically have random XCI patterns [55, 56]. Hence, patients with RTT have a mosaic distribution of cells expressing the wild-type MECP2 and cells expressing the mutant MECP2 allele in all of their tissues. Analysis of gene expression profiles on tissues from deceased girls with RTT will therefore be compromised by unpredictable patterns of XCI. In addition, the presence of functional "wild-type" cells will also dampen small effects on the expression levels of MeCP2 target genes. Postmortem tissues are mostly from girls at later stages of the disorder and their gene expression profiles may reflect the presence of concurrent disease.
Because central nervous system neurons are the primary cell types where the RTT syndrome pathology occurs, a pure population of neurons with the mutant MECP2 on the active X chromosome, compared to one with the wild-type MECP2 on the active X chromosome from the same individual would constitute an ideal sample set for analysis of gene expression profiles in the presence of MECP2 mutations, but such samples are unavailable from human patients and alternative strategies are needed.
MECP2 is expressed, albeit at lower levels, in nearly all other tissues, including T-lymphocytes. Although T-lymphocytes are not the primary cell type where the RTT phenotype manifests, previous experiments have shown that lymphocytes with the mutant MECP2 on the active X chromosome are at a growth disadvantage compared to those with the wild-type MECP2 on the active X chromosome . This suggested to us that at least a subset of the MeCP2 target genes are dysregulated in this cell type and pure T-lymphocyte clonal cell lines may be a valuable source of RNA for gene expression profiling in Rett syndrome.
We isolated single T-lymphocytes from blood samples of RTT patients and clonally expanded them in order to compare MECP2 mutant and wild type expression in the same patient. Since only one form of MECP2 would be expressed in each isolated clone, we expected that effects on gene expression profiles would be more pronounced. Furthermore, by comparing these clonal T-lymphocytes from the same patient, we could eliminate changes resulting from interindividual genetic differences. A similar approach was taken by Traynor et al., 2002, but instead of lymphocytes they used lymphoblastoid cell lines and fibroblast clones . Lymphoblastoid cells have the disadvantage that they are immortalized which can affect gene expression patterns, while fibroblast cultures can gain epigenetic differences as they divide in culture. Although the T-lymphocytes used in our experiment were also cultured, the number of cell divisions was small and the cells were not transformed.
Because we did not study the primary cell type that is responsible for the RTT pathology, we strove to increase the likelihood that any observed differences in gene expression levels between mutant and wild-type MECP2-expressing clones were relevant to Rett syndrome. We used samples from patients with two different types of mutations (p.T158M and c.1308-1309delTC), combined the results from these samples to focus only on genes with a consistent pattern of increased or decreased levels of expression in all mutant clones compared to wild type. We next compared these results to those of our other independent microarray experiment on Mecp2-mutant mouse EBs and to those published in the literature from expression profiling [29–31, 44] or large-scale ChIP experiments . The limited overlap in identified genes with up or downregulated expression between all these different lists cannot be easily explained, but could have a number of different reasons. The tissues, or cell lines studied and specific mutations varied between the studies. Human as well as mouse samples were analyzed and they originated from different stages of the disease process. More likely reasons may be that overall expression differences between mutant and wild-type MeCP2 RNA samples are subtle but affect a large number of genes. An alternate hypothesis is that MeCP2 primarily regulates expression of a limited number of genes in very specific cells or in a transient manner. Such changes would be difficult to detect with the strategies used in any of these expression profiling studies.
One of the genes identified by our comparative analysis was SPOCK1. Its mouse homologue encodes Spock1/testican, a Ca(2+)-binding proteoglycan predominantly located in the extracellular matrix. Although its function is incompletely understood, Spock1/testican contains six functional domains, several of which have a protease-inhibitor function . There is an N-terminal hydrophobic signal sequence for secretion of the protein in the extracellular matrix, a cysteine-rich region, a Kazal-like homology domain with a putative seine-protease inhibitor role, comprised in several regions of homology to SPARC/osteonectin, two calcium-binding EF hand motifs, and a C-terminal domain with homology to thyrotropin cysteine protease inhibitors (CVCW or thyorglobulin-like domain) that has been shown to inhibit the lysosomal cysteine protease cathepsin-L . Spock1 is highly enriched in brain and is associated with the postsynaptic region of a subpopulation of pyramidal neurons in the CA3 area of the hippocampus . It is also found in the choroid plexus and is highly enriched in the central nervous system (CNS) during neuronal proliferation and migration . It affects neuronal attachment and neurite outgrowth likely through a modulation of the extracellular matrix . Quantitative RT-PCR experiments on the clonal lymphocyte cultures showed only a trend for downregulation. Quantitative RT-PCR experiments on brain regions of Mecp2 mutant mice also did not show consistent changes in expression. This suggests that SPOCK1 is not a direct target of MeCP2 and its downregulation in the microarray experiment might have been a false positive finding.
To find new putative MeCP2 targets, we performed expression profiling on clonal lymphocyte cultures from RTT patients with two different MECP2 mutations and compared the gene expression profiles to those of other screens for MeCP2 targets. No statistically significant differences in expression were found upon stringent analysis of the data. A putative downregulated MeCP2-target gene, SPOCK1, did not have altered expression in brain regions in a mouse model of Rett syndrome.
This work was supported by a grant from the National Institutes of Health (NIH P01-HD40301) and the Microarray Expression Core of the Baylor Mental Retardation and Developmental Disabilities Research Center (NIH HD24064). The authors thank Huda Zoghbi for sharing the Mecp2 R308 mutant mice and Miguel Landers for valuable input during preparation of this manuscript.
- Akesson HO, Hagberg B, Wahlstrom J: Rett syndrome, classical and atypical: genealogical support for common origin. J Med Genet. 1996, 33 (9): 764-766.View ArticlePubMedPubMed CentralGoogle Scholar
- Hagberg B, Hagberg G: Rett syndrome: epidemiology and geographical variability. Eur Child Adolesc Psychiatry. 1997, 6 (Suppl 1): 5-7.PubMedGoogle Scholar
- Armstrong DD: Review of Rett syndrome. J Neuropathol Exp Neurol. 1997, 56 (8): 843-849.View ArticlePubMedGoogle Scholar
- Weaving LS, Ellaway CJ, Gecz J, Christodoulou J: Rett syndrome: clinical review and genetic update. J Med Genet. 2005, 42 (1): 1-7. 10.1136/jmg.2004.027730.View ArticlePubMedPubMed CentralGoogle Scholar
- 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. 10.1038/13810.View ArticlePubMedGoogle Scholar
- Miltenberger-Miltenyi G, Laccone F: Mutations and polymorphisms in the human methyl CpG-binding protein MECP2. Hum Mutat. 2003, 22 (2): 107-115. 10.1002/humu.10243.View ArticlePubMedGoogle Scholar
- Van den Veyver IB, Zoghbi HY: Methyl-CpG-binding protein 2 mutations in Rett syndrome. Curr Opin Genet Dev. 2000, 10 (3): 275-279. 10.1016/S0959-437X(00)00083-6.View ArticlePubMedGoogle Scholar
- Van den Veyver IB, Zoghbi HY: Genetic basis of Rett syndrome. Ment Retard Dev Disabil Res Rev. 2002, 8 (2): 82-86. 10.1002/mrdd.10025.View ArticlePubMedGoogle Scholar
- Amir RE, Fang P, Yu Z, Glaze DG, Percy AK, Zoghbi HY, Roa BB, Van den Veyver IB: Mutations in exon 1 of MECP2 are a rare cause of Rett Syndrome. J Med Genet. 2005, 42 (2): e15-10.1136/jmg.2004.026161.View ArticlePubMedPubMed CentralGoogle Scholar
- Laccone F, Junemann I, Whatley S, Morgan R, Butler R, Huppke P, Ravine D: Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome. Hum Mutat. 2004, 23 (3): 234-244. 10.1002/humu.20004.View ArticlePubMedGoogle Scholar
- Archer HL, Whatley SD, Evans JC, Ravine D, Huppke P, Kerr A, Bunyan D, Kerr B, Sweeney E, Davies SJ, Reardon W, Horn J, Macdermot KD, Smith RA, Magee A, Donaldson A, Crow Y, Hermon G, Miedzybrodzka Z, Cooper DN, Lazarou L, Butler R, Sampson J, Pilz DT, Laccone F, Clarke AJ: Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients. J Med Genet. 2006, 43 (5): 451-456. 10.1136/jmg.2005.033464.View ArticlePubMedGoogle Scholar
- Shi J, Shibayama A, Liu Q, Nguyen VQ, Feng J, Santos M, Temudo T, Maciel P, Sommer SS: Detection of heterozygous deletions and duplications in the MECP2 gene in Rett syndrome by Robust Dosage PCR (RD-PCR). Hum Mutat. 2005, 25 (5): 505-10.1002/humu.9338.View ArticlePubMedGoogle Scholar
- Kriaucionis S, Bird A: The major form of MeCP2 has a novel N-terminus generated by alternative splicing. Nucleic Acids Res. 2004, 32 (5): 1818-1823. 10.1093/nar/gkh349.View ArticlePubMedPubMed CentralGoogle Scholar
- Mnatzakanian GN, Lohi H, Munteanu I, Alfred SE, Yamada T, MacLeod PJ, Jones JR, Scherer SW, Schanen NC, Friez MJ, Vincent JB, Minassian BA: A previously unidentified MECP2 open reading frame defines a new protein isoform relevant to Rett syndrome. Nat Genet. 2004, 36 (4): 339-341. 10.1038/ng1327.View ArticlePubMedGoogle Scholar
- Philippe C, Villard L, De Roux N, Raynaud M, Bonnefond JP, Pasquier L, Lesca G, Mancini J, Jonveaux P, Moncla A, Chelly J, Bienvenu T: Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update. Eur J Med Genet. 2006, 49 (1): 9-18. 10.1016/j.ejmg.2005.04.003.View ArticlePubMedGoogle Scholar
- Evans JC, Archer HL, Whatley SD, Kerr A, Clarke A, Butler R: Variation in exon 1 coding region and promoter of MECP2 in Rett syndrome and controls. Eur J Hum Genet. 2005, 13 (1): 124-126. 10.1038/sj.ejhg.5201270.View ArticlePubMedGoogle Scholar
- Nan X, Meehan RR, Bird A: Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Res. 1993, 21 (21): 4886-4892.View ArticlePubMedPubMed CentralGoogle Scholar
- Nan X, Campoy FJ, Bird A: MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell. 1997, 88 (4): 471-481. 10.1016/S0092-8674(00)81887-5.View ArticlePubMedGoogle Scholar
- 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. 10.1038/561.View ArticlePubMedGoogle Scholar
- Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A: Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature. 1998, 393 (6683): 386-389. 10.1038/30764.View ArticlePubMedGoogle Scholar
- Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T: The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem. 2003, 278 (6): 4035-4040. 10.1074/jbc.M210256200.View ArticlePubMedGoogle Scholar
- Kimura H, Shiota K: Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA methyltransferase, Dnmt1. J Biol Chem. 2003, 278 (7): 4806-4812. 10.1074/jbc.M209923200.View ArticlePubMedGoogle Scholar
- Harikrishnan KN, Chow MZ, Baker EK, Pal S, Bassal S, Brasacchio D, Wang L, Craig JM, Jones PL, Sif S, El-Osta A: Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing. Nat Genet. 2005, 37 (3): 254-264. 10.1038/ng1516.View ArticlePubMedGoogle Scholar
- Young JI, Hong EP, Castle JC, Crespo-Barreto J, Bowman AB, Rose MF, Kang D, Richman R, Johnson JM, Berget S, Zoghbi HY: Regulation of RNA splicing by the methylation-dependent transcriptional repressor methyl-CpG binding protein 2. Proc Natl Acad Sci USA. 2005, 102 (49): 17551-17558. 10.1073/pnas.0507856102.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R, Greenberg ME: Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science. 2003, 302 (5646): 885-889. 10.1126/science.1086446.View ArticlePubMedGoogle Scholar
- Martinowich K, Hattori D, Wu H, Fouse S, He F, Hu Y, Fan G, Sun YE: DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science. 2003, 302 (5646): 890-893. 10.1126/science.1090842.View ArticlePubMedGoogle Scholar
- Chang Q, Khare G, Dani V, Nelson S, Jaenisch R: The disease progression of Mecp2 mutant mice is affected by the level of BDNF expression. Neuron. 2006, 49 (3): 341-348. 10.1016/j.neuron.2005.12.027.View ArticlePubMedGoogle Scholar
- Stancheva I, Collins AL, Van den Veyver IB, Zoghbi H, Meehan RR: A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos. Mol Cell. 2003, 12 (2): 425-435. 10.1016/S1097-2765(03)00276-4.View ArticlePubMedGoogle Scholar
- Traynor J, Agarwal P, Lazzeroni L, Francke U: Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations. BMC Med Genet. 2002, 3: 12-10.1186/1471-2350-3-12.View ArticlePubMedPubMed CentralGoogle Scholar
- Tudor M, Akbarian S, Chen RZ, Jaenisch R: Transcriptional profiling of a mouse model for Rett syndrome reveals subtle transcriptional changes in the brain. Proc Natl Acad Sci USA. 2002, 99 (24): 15536-15541. 10.1073/pnas.242566899.View ArticlePubMedPubMed CentralGoogle Scholar
- Colantuoni C, Jeon OH, Hyder K, Chenchik A, Khimani AH, Narayanan V, Hoffman EP, Kaufmann WE, Naidu S, Pevsner J: Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification. Neurobiol Dis. 2001, 8 (5): 847-865. 10.1006/nbdi.2001.0428.View ArticlePubMedGoogle Scholar
- Pescucci C, Meloni I, Renieri A: Is Rett syndrome a loss-of-imprinting disorder?. Nat Genet. 2005, 37 (1): 10-11. 10.1038/ng0105-10.View ArticlePubMedGoogle Scholar
- Klose R, Bird A: Molecular biology. MeCP2 repression goes nonglobal. Science. 2003, 302 (5646): 793-795. 10.1126/science.1091762.View ArticlePubMedGoogle Scholar
- Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu T: Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nat Genet. 2005, 37 (1): 31-40.PubMedGoogle Scholar
- Balmer D, LaSalle JM: Clonal maintenance of imprinted expression of SNRPN and IPW in normal lymphocytes: correlation with allele-specific methylation of SNRPN intron 1 but not intron 7. Hum Genet. 2001, 108 (2): 116-122. 10.1007/s004390000455.View ArticlePubMedGoogle Scholar
- LaSalle JM, Ritchie RJ, Glatt H, Lalande M: Clonal heterogeneity at allelic methylation sites diagnostic for Prader-Willi and Angelman syndromes. Proc Natl Acad Sci USA. 1998, 95 (4): 1675-1680. 10.1073/pnas.95.4.1675.View ArticlePubMedPubMed CentralGoogle Scholar
- Balmer D, Arredondo J, Samaco RC, LaSalle JM: MECP2 mutations in Rett syndrome adversely affect lymphocyte growth, but do not affect imprinted gene expression in blood or brain. Hum Genet. 2002, 110 (6): 545-552. 10.1007/s00439-002-0724-4.View ArticlePubMedGoogle Scholar
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5 (10): R80-10.1186/gb-2004-5-10-r80.View ArticlePubMedPubMed CentralGoogle Scholar
- Bioconductor. [http://www.bioconductor.org]
- Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003, 4 (2): 249-264. 10.1093/biostatistics/4.2.249.View ArticlePubMedGoogle Scholar
- Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003, 19 (2): 185-193. 10.1093/bioinformatics/19.2.185.View ArticlePubMedGoogle Scholar
- Wu Z, Irizarry RA, Gentleman R, Martinez Murillo F, Spencer F: A model based background adjustment for oligonucleotide expression arrays. Johns Hopkins Dept of Biostatistics Working Papers. 2004, (paper 1).Google Scholar
- Shahbazian MD, Young YI, Yuva-Paylor LA, Spencer CM, Antalffy BA, Noebels JL, Armstrong DL, Paylor R, Zoghbi HY: Mice with Truncated MeCP2 Recapitulate Many Rett Syndrome Features and Display Hyperacetylation of Histone H3. Neuron. 2002, 35: 243-254. 10.1016/S0896-6273(02)00768-7.View ArticlePubMedGoogle Scholar
- Ballestar E, Ropero S, Alaminos M, Armstrong J, Setien F, Agrelo R, Fraga MF, Herranz M, Avila S, Pineda M, Monros E, Esteller M: The impact of MECP2 mutations in the expression patterns of Rett syndrome patients. Hum Genet. 2005, 116 (1–2): 91-104. 10.1007/s00439-004-1200-0.View ArticlePubMedGoogle Scholar
- National Center for Biotechnoology Information Entrez Gene. [http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/entrez/query.fcgi?db=gene]
- Applied Biosystems user bulletin #2: ABI PRISM 7700 Sequence Detection System. [http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf]
- Alvarez E, Zhou W, Witta SE, Freed CR: Characterization of the Bex gene family in humans, mice, and rats. Gene. 2005, 357 (1): 18-28. 10.1016/j.gene.2005.05.012.View ArticlePubMedGoogle Scholar
- Phan J, Reue K: Lipin, a lipodystrophy and obesity gene. Cell Metab. 2005, 1 (1): 73-83. 10.1016/j.cmet.2004.12.002.View ArticlePubMedGoogle Scholar
- Suviolahti E, Reue K, Cantor RM, Phan J, Gentile M, Naukkarinen J, Soro-Paavonen A, Oksanen L, Kaprio J, Rissanen A, Salomaa V, Kontula K, Taskinen MR, Pajukanta P, Peltonen L: Cross-species analyses implicate Lipin 1 involvement in human glucose metabolism. Hum Mol Genet. 2006, 15 (3): 377-386. 10.1093/hmg/ddi448.View ArticlePubMedGoogle Scholar
- Alliel PM, Perin JP, Jolles P, Bonnet FJ: Testican, a multidomain testicular proteoglycan resembling modulators of cell social behaviour. Eur J Biochem. 1993, 214 (1): 347-350. 10.1111/j.1432-1033.1993.tb17930.x.View ArticlePubMedGoogle Scholar
- Marr HS, Basalamah MA, Bouldin TW, Duncan AW, Edgell CJ: Distribution of testican expression in human brain. Cell Tissue Res. 2000, 302 (2): 139-144. 10.1007/s004410000277.View ArticlePubMedGoogle Scholar
- Charbonnier F, Chanoine C, Cifuentes-Diaz C, Gallien CL, Rieger F, Alliel PM, Perin JP: Expression of the proteoglycan SPOCK during mouse embryo development. Mech Dev. 2000, 90 (2): 317-321. 10.1016/S0925-4773(99)00255-5.View ArticlePubMedGoogle Scholar
- Marr HS, Edgell CS: Testican-1 inhibits attachment of Neuro-2a cells. Matrix Biology. 2003, 22: 259-266. 10.1016/S0945-053X(03)00036-2.View ArticlePubMedGoogle Scholar
- Bonnet F, Perin JP, Charbonnier F, Camuzat A, Roussel G, Nussbaum JL, Alliel PM: Structure and cellular distribution of mouse brain testican. Association with the postsynaptic area of hippocampus pyramidal cells. J Biol Chem. 1996, 271 (8): 4373-4380. 10.1074/jbc.271.8.4373.View ArticlePubMedGoogle Scholar
- Shahbazian MD, Sun Y, Zoghbi HY: Balanced X chromosome inactivation patterns in the Rett syndrome brain. Am J Med Genet. 2002, 111 (2): 164-168. 10.1002/ajmg.10557.View ArticlePubMedGoogle Scholar
- Amir RE, Zoghbi HY: Rett syndrome: methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations. Am J Med Genet. 2000, 97 (2): 147-152. 10.1002/1096-8628(200022)97:2<147::AID-AJMG6>3.0.CO;2-O.View ArticlePubMedGoogle Scholar
- BaSalamah MA, Marr HS, Duncan AW, Edgell CJ: Testican in human blood. Biochem Biophys Res Commun. 2001, 283 (5): 1083-1090. 10.1006/bbrc.2001.4900.View ArticlePubMedGoogle Scholar
- Bocock JP, Edgell CJ, Marr HS, Erickson AH: Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L. Eur J Biochem. 2003, 270 (19): 4008-4015. 10.1046/j.1432-1033.2003.03789.x.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2350/7/61/prepub
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