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Mecp2 Knockdown Reveals DNA Methylation-Independent Gene Repression of Target Genes in Living Cells and a Bias in the Cellular Location of Target Gene Products

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Mecp2 Knockdown Reveals DNA Methylation-Independent Gene Repression of Target Genes in Living Cells and a Bias in the Cellular Location of Target Gene Products Genes Genet. Syst. (2008) 83, p. 199–208 MeCP2 knockdown reveals DNA methylation-independent gene repression of target genes in living cells and a bias in the cellular location of target gene products Shinya Yakabe1,2, Hidenobu Soejima1*, Hitomi Yatsuki1, Hirotaka Tominaga3, Wei Zhao4, Ken Higashimoto1, Keiichiro Joh1, Shinichi Kudo5, Kohji Miyazaki2 and Tsunehiro Mukai6 1Division of Molecular Genetics and Epigenetics, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan 2Division of General Surgery, Department of Surgery, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan 3Section of Clinical Cooperation System, Center for Comprehensive Community Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan 4Department of Cardiovascular Medicine, Shanghai Shuguang Hospital Affiliated with Shanghai University of T.C.M., No. 185 Pu An Road, Shanghai 200021, R. P. China 5Hokkaido Institute of Public Health, Sapporo 060-0819, Japan 6Saga University, 1 Honjo, Saga, Japan (Received 9 February 2008, accepted 21 February 2008) MeCP2, a methyl-CpG binding domain (MBD) protein, is known to bind to meth- ylated CpG sites via a conserved MBD, leading to transcriptional repression. However, studies in cell-free system for gene repression and MeCP2 binding have suggested that DNA methylation-independent repression also occurs in living cells. It has been difficult to characterize the target genes of MeCP2 because a limited number have been identified to date. In this context, we screened for MeCP2 target genes using knockdown (KD) experiments combined with microar- ray gene expression analyses. Of the 49 genes that showed a more than three- fold increase in expression in two independent KD experiments conducted with different siRNA sets, unexpectedly, half (24 genes) did not contain promoter CpG islands (CGIs). Of seven selected genes that did contain CGIs, only two were methylated at the CGI, bound MeCP2 before KD, and reduced MeCP2 after KD. For three, MeCP2 was observed to bind to the unmethylated CGI before KD, and for one MeCP2 was reduced after KD. Another two genes neither had DNA meth- ylation nor bound MeCP2 before KD. Gene ontology analysis suggested that MeCP2 represses a certain group of genes. These results suggest that in addition to the canonical gene repression function, MeCP2 can repress gene expression by binding to unmethylated DNA in particular genes in living cells. Key words: CpG island, DNA methylation, gene ontology, gene repression, MeCP2 2003). In human cancers, a number of genes are known INTRODUCTION to be repressed by epigenetic means, for example aberrant Epigenetic regulation of gene expression plays a critical hypermethylation of the promoter CpG island (CGI). role in development and differentiation, X inactivation, One way in which DNA hypermethylation represses gene and genomic imprinting; it also plays a role in several expression may be inhibition of the binding of transcrip- human diseases, including cancer (Jaenisch and Bird, tion factors to their target sequences. However, it is also widely accepted that methylated CpGs are recognized by Edited by Hiroshi Nojima the methyl-CpG binding proteins, including MeCP2, * Corresponding author. E-mail: [email protected] MBD1, MBD2, MBD3, MBD4 and Kaiso, and that these 200 S. YAKABE et al. proteins recruit protein complexes related to histone mod- (Invitrogen) according to the manufacturer’s protocol. ification and chromatin remodeling (Jaenisch and Bird, The dsRNA sets were as follows: Set A, synthesized 2003; Bienvenu and Chelly, 2006). Recent studies have dsRNA with sequences 5’-CCUAAUGAUUUUGACUU- shown that of these, only MeCP2, MBD1 and MBD2 act CACGGUACAG-3’ and 5’-UUACCGUGAAGUCAAAAU- as methyl-CpG binding proteins in mammals (Lopez- CAUUAGGAU-3’ (Hokkaido System Science, Sapporo, Serra et al., 2006). Japan); Set B, dsRNAs (Stealth Select RNAi) of three dif- MeCP2 is a member of the methyl-CpG binding domain ferent sequences (Invitrogen, Catalog #1299003). A single (MBD) family and plays a pivotal role in DNA methyla- nucleotide at the 5’ terminal of the Set A antisense tion-associated gene repression. MECP2 is a causative sequence was intentionally mismatched to improve the gene for Rett syndrome, a dominant X-linked neurodevel- knockdown effect of the siRNA (Schwarz et al., 2003). At opmental disorder in which affected individuals are usu- 72 hours after transfection, cells were harvested for use ally heterozygous for a de novo mutation in MECP2 in further experiments. (Bienvenu and Chelly, 2006). The canonical gene repres- sion function of MeCP2 involves the molecule binding to RNA extraction and reverse transcription PCR methylated CpG sites via a conserved MBD, leading to Total RNA was extracted using an RNeasy mini kit transcriptional repression, which occurs due to recruit- (Qiagen, Hilden, Germany) with an RNase-free DNase kit ment of Sin3A and histone deacetylases (HDACs) and/or (Qiagen). Total RNA (500 ng) was reverse-transcribed mediation of the methylation of histone H3 lysine 9 with random primers using ReverTra Ace reverse tran- (Bienvenu and Chelly, 2006; Fuks et al., 2003a; Fuks et scriptase (Toyobo, Osaka, Japan). Gene expression was al., 2003b). In cell-free system, MeCP2 can bind to unm- quantitated by real-time PCR on an ABI Prism 7000 with ethylated nucleosomal arrays and repress transcription TaqMan probe (Applied Biosystems, Foster City, CA, from both methylated and unmethylated naked DNA USA) and QuantiTect SYBR Green PCR kits (Qiagen) for (Meehan et al., 1992; Nan et al., 1997; Kaludov and MBDs and other genes, respectively, as shown in Table Wolffe, 2000; Georgel et al., 2003). This naturally sug- 1. The expression level of each gene was normalized gests that MeCP2 may be able to repress gene expression against that of the housekeeping genes GAPDH or β- without DNA methylation in living cells; however, this actin. All quantitative RT-PCRs were performed in trip- hypothesis has not been tested. Given that mutations in licate. MECP2 cause Rett syndrome, attempts have been made to identify the target genes of MeCP2 in the brain, with Western blotting Proteins were extracted from LU65 the result that a limited number of target genes have cells with and without MECP2 KD using SDS lysis buffer been identified (Bienvenu and Chelly, 2006). However, (2% SDS, 50 mM Tris-HCl, pH 7.5). Aliquots (5 μg) of no attempt has been made to perform a genome-wide protein were loaded onto 10% SDS-PAGE gels and elec- screen for MeCP2 target genes in cancer cells, and it trophoresed, then blotted onto PVDF membranes using a remains unknown whether MeCP2 regulates gene expres- semi-dry blotting method. The membranes were probed sion without DNA methylation in living cells. Thus, in with antibodies against MeCP2 (Kudo, 1998), MBD1 this study we screened potential MeCP2 target genes (Abcam, Cambridge, UK, cat. 3753 or 2846), MBD2/3 using knockdown (KD) with siRNA and microarray gene (Millipore, Billerica, MA, USA, cat. 07-199), and β-actin expression analyses, and investigated DNA methylation (Sigma, cat. A5441). An ECL plus western blotting and the binding of MeCP2 to CGIs within promoter regions. We also performed gene ontology (GO) analysis Table 1. Primers for quantitative RT-PCRs for the identified MeCP2 target genes in an effort to Gene name Catalogue number Detection kit determine if they have any features in common. MeCP2 Applied Biosystems, Hs00172845_m1 TaqMan MBD1 Applied Biosystems, Hs00242770_m1 TaqMan MATERIALS AND METHODS MBD2 Applied Biosystems, Hs00187506_m1 TaqMan Cell Lines LU65 cells derived from human lung cancer GAPDH Applied Biosystems, Hs99999905_m1 TaqMan and BEAS-2B cells from human bronchial epithelium DNER Qiagen, QT00045955 SYBR GREEN were cultured in RPMI-1640 medium (Sigma, St. Louis, HAS3 Qiagen, QT00014903 SYBR GREEN MO, USA) supplemented with 10% FCS (Gibco, Invitrogen, IL6R Qiagen, QT00023660 SYBR GREEN Carlsbad, CA, USA) and in MEM-α medium (Gibco) sup- CYP1A1 Qiagen, QT00012341 SYBR GREEN plemented with 10% FCS, respectively. Cells were har- ST3GAL5 Qiagen, QT00054236 SYBR GREEN vested at 70% confluence. FOXA1 Qiagen, QT00212828 SYBR GREEN RNAi experiments We transfected LU65 cells with IFIH1 Qiagen, QT00033789 SYBR GREEN two different dsRNA sets using Lipofectamine 2000 β-actin Qiagen, QT00095431 SYBR GREEN Characterization of MeCP2 target genes via knockdown 201 detection system (GE Healthcare, Buckinghamshire, UK) DNA methylation analyses Genomic DNA extracted was used for detection. from LU65 cells with and without MECP2 KD was sub- jected to sodium bisulfite modification with an EpiTect Microarray expression analysis Total RNA (500 ng) bisulfite kit (Qiagen). Modified DNA was amplified by from LU65 cells with and without MECP2 KD was ampli- PCR with a primer set specific to each gene, followed by fied using a Low RNA Fluorescent Linear Amplification cloning and sequencing. DNA methylation status in Kit (Agilent Technologies, Santa Clara, CA, USA) and LU65 and BEAS-2B cells was also analyzed by combined labeled with Cy-5 and Cy-3, respectively. Labeled cRNAs bisulfite restriction analysis (COBRA). All primers used were co-hybridized with the Whole Human Genome Oligo in this study are shown in Table 2. Microarray (Agilent Technologies), which includes 41,000 human genes and transcripts. Data were extracted Chromatin immunoprecipitation (ChIP) Chro- from the resulting images using Agilent’s Feature
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