Human Molecular Genetics, 2001, Vol. 10, No. 10 1085-1092
© 2001 Oxford University Press
MECP2 truncating mutations cause histone H4 hyperacetylation in Rett syndrome
1Department of Genetics and 2Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA and 3Laboratory of Molecular Immunology, National Heart Lung and Blood Institute, NIH, Bethesda, MD, USA
Received 29 January 2001; Revised and Accepted 15 March 2001.
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ABSTRACT |
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Rett syndrome (RTT) is a mostly sporadic disorder of developmental regression, with loss of speech and purposeful hand use, microcephaly and seizures. It affects 1 in 10 000–15 000 females. RTT is caused by mutations in the MECP2 gene, which is located in Xq28 and subject to X inactivation. MECP2 encodes a methyl-CpG-binding protein that binds to 5-methyl-cytosine in DNA through its methyl-binding domain. Recruitment of a transcriptional silencing complex through MeCP2’s transcriptional repression domain results in histone deacetylation and chromatin condensation. To study the effects of two common truncating RTT mutations (R168X and 803delG), we examined mutant MeCP2 expression and global histone acetylation levels in clonal cell cultures from a female RTT patient with the mutant R168X allele on the active X chromosome, as well as in cells from a male hemizygous for the frameshift mutation 803delG (V288X). Both mutant alleles generated stable RNA transcripts, but no intact MeCP2 protein was detected with an antibody against the C-terminal region of MeCP2. Western blots with antibodies against acetylated histones H3 and H4 revealed that H4, but not H3, was hyperacetylated. By using antibodies against individual acetylated lysine residues, the observed H4 hyperacetylation was attributed to increased acetylation of lysine 16. Therefore, expression of endogenous truncating MECP2 alleles, in the absence of wild-type MeCP2 protein, is specifically associated with an increase in the mono-acetylated histone isoform H4K16. This observed effect may result in over-expression of MeCP2 target genes and, thus, play a role in the pathogenesis of RTT.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Rett syndrome (RTT) is a progressive neurological disorder and one of the most common causes of mental retardation in females (1). RTT is characterized by apparently normal early postnatal development followed by developmental arrest and loss of cognitive functions (2,3). Girls with classic RTT appear to be normal at birth and develop normally until 6–18 months of age, then gradually lose speech and purposeful hand use. They develop stereotypic hand movements, deceleration of head growth, seizures, gait apraxia, intermittent hyperventilation, autistic behavior and mental retardation. Sudden unexplained death may occur. Loss of ambulation and development of scoliosis accompany survival to adulthood. While the majority of RTT cases are sporadic, a few families with multiple affected females, related through females, suggested a genetic defect. Studies of rare familial cases led to the hypothesis that RTT is caused by X-linked dominant mutations in a gene subject to X chromosome inactivation (XCI). Exclusion mapping of the putative X-linked RTT gene in these families localized the gene to Xq28 (4–8). By systematic mutation analysis of positional candidate genes in Xq28, RTT-causing mutations were identified in the MECP2 gene encoding a methyl-CpG-binding protein (MeCP2) (9,10).
MeCP2 is an abundant, ubiquitously expressed DNA-binding protein which is thought to act as a global transcriptional repressor (11,12). It contains two important functional domains; the 85-amino acid methyl-CpG binding domain (MBD) (13) and the 102-amino acid transcriptional repression domain (TRD) (11). MeCP2 binds specifically to 5-methylcytosine through its MBD and the TRD recruits a transcriptional silencing complex resulting in histone deacetylation and chromatin condensation (12,14). Although histone deacetylase (HDAC)-independent pathways for transcriptional repression by MeCP2 have recently been defined by in vivo and in vitro studies (15,16), there is strong evidence for a HDAC-dependent pathway for transcriptional repression mediated by the TRD of MeCP2 (12,14). In the HDAC-dependent pathway, MeCP2 interacts with the corepressor Sin3A to recruit HDAC1 and 2, which in turn results in deacetylation of core histones and transcriptional silencing (12,14). MeCP2 binds, with comparable affinity, to a single symmetrically methylated CpG both in naked and nucleosomal DNA (17,18). Because the 2 bp binding site of MeCP2 occurs once per 150 bp throughout most of the vertebrate genomes, deficiency of MeCP2 may cause global histone hyperacetylation.
Histone acetylation is an important regulator of transcription in eukaryotic organisms (19). Multiple histone acetyltransferases (HATs) and HDACs maintain a dynamic equilibrium of histone acetylation (19,20). Several transcriptional activator or co-activator complexes possessing HAT activity are required for transcriptional activation, whereas co-repressor complexes containing HDACs are linked to transcriptional repression (21). In general, histone acetylation is associated with transcriptionally active, or potentially active, chromatin, whereas hypoacetylated histones are found in transcriptionally inactive or heterochromatin (22). Among the core histones, H3 and H4 are the principal targets of regulatory post-transcriptional modification, such as HDAC enzymatic activity. The N-termini of these two core histones are remarkably conserved across species, and each contains multiple conserved lysine (K) residues that serve as acetylation sites (23). In histone H4, acetylation occurs at K5, K8, K12 and K16, whereas acetylation of histone H3 occurs at K9, K14, K18 and K23. Acetylation of different histones and at specific K residues can have differential effects on target gene expression (24) (see Discussion for examples).
Here we report the biochemical effects of two common truncating mutations (R168X and 803delG) found in RTT patients (10). To test the hypothesis that truncating mutations abolishing the TRD of MeCP2 cause histone hyperacetylation in RTT cells, we examined MeCP2 expression and global histone acetylation levels in clonal cell cultures from a female RTT patient with the mutant R168X allele on the active X chromosome, as well as in cells from a male hemizygous for 803delG (10). In both cell types, we found hyperacetylation of histone H4, but not H3, which occurred predominantly at K16.
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RESULTS |
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Establishment of clonal cell lines with the mutant MECP2 allele on the active X chromosome
Females with RTT have random X inactivation patterns (25,26). Therefore, tissues from these individuals are mosaic, consisting of a mixture of cells with either the normal or the mutant MECP2 allele on the active X. To study the effects of MECP2 mutations on gene expression and chromatin organization, we set out to establish clonal cell lines with either one or the other X chromosome active. Lymphoblastoid cell lines from two affected sisters who had inherited an R168X mutation from their carrier mother were chosen as parental cell lines (8,10). The 502C
T transition causing a stop codon (R168X) between the MBD and the TRD was first reported in seven independent families by us (10). It is the most common recurring MECP2 mutation, having been identified in 15% of RTT patients from around the world (27). The XCI status of the clonal cell lines was assessed by the widely used androgen receptor (AR) assay, which is based on differential methylation of cytosine residues in HpaII restriction sites, located just 5' of a highly polymorphic CAG repeat in the first exon of the AR gene (28). On the inactive X chromosome, the HpaII sites are methylated and resistant to HpaII digestion, whereas on the active X chromosome, the sites are unmethylated and susceptible to HpaII cleavage. In this family, the PCR fragments representing the paternal AR allele are migrating more slowly than the ones representing the maternal allele (Fig. 1A). In cell line 1 derived from one of the affected sisters, the paternal chromosome carrying the normal MECP2 allele is the active one, whereas in clonal lines 2 and 3 derived from the other sister, the maternal AR allele is subject to HpaII digestion, indicating that the X chromosome carrying the mutant MECP2 gene is active. For further studies, cell line 1 was used as a normal control for comparison with the mutant cell lines 2 and 3.
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In addition, a lymphoblastoid cell line from a 46,XY male hemizygote who inherited a MECP2 frameshift mutation, 803delG, leading to a stop codon (V288X) within the TRD, was studied in comparison with a normal male lymphoblastoid cell line. Whereas this mutant male was affected with a severe congenital encephalopathy, the same mutation caused classic RTT in his sister and maternal aunt (7,10). Independent recurrences of 803delG in other RTT females have been reported (27).
Mutant mRNA is present and intact MeCP2 protein is absent in the mutant cell lines
The expression of the mutant MECP2 alleles was first assessed at the RNA level by RT–PCR. The R168X mutant transcript was distinguished from the transcript of the normal allele by HphI restriction analysis of the RT–PCR products. The 502CT transition creates a new cleavage site for HphI (10). RT–PCR products from cell lines 2 and 3, but not from cell line 1, were completely digested by HphI (Fig. 1B). Therefore, cell lines 2 and 3 exclusively express the mutant R168X allele, whereas cell line 1 expresses the wild-type MECP2 allele. RT–PCR products of the mutant allele appeared to be of equal abundance to those of the normal allele on agarose gels, although accurate quantitation was not performed. As predicted, the mutant mRNA containing a stop codon in the 3'-most exon is not subject to nonsense-mediated mRNA decay. Likewise, RT–PCR analysis of the male 803delG cell line suggested stability of the mutant mRNA (Fig. 1C).
On western blot analysis with an antibody against the C-terminal region of MeCP2, a reacting protein band was seen only in cell lines expressing a normal MECP2 gene (samples 1 and 5; Fig. 1B). Given the location of the antigenic site, this antibody would not detect the putative truncated proteins derived from the mutant alleles in cell lines 2, 3 and 4. As we elaborated previously (10), the putative 167-amino acid truncated product of the R168X allele lacks the nuclear localization signal and would be expected to remain in the cytoplasm. Therefore, it is unlikely that this truncated protein could bind to nuclear methyl-CpG and exert a dominant negative effect. Given that both truncation mutations studied here cause the same phenotype, we assume that both lead to loss of function. The absence of the normal-sized MeCP2 protein in the female clonal cell lines 2 and 3 conveys two important messages. Firstly, it confirms that these cell lines are truly pure with respect to their active X chromosome, as was suggested by the AR (XCI) assay and RT–PCR studies. Secondly, the absence of functional MeCP2 protein in these two mutant cell lines has apparently not led to reactivation of the normal MECP2 allele, located on the inactive X, during the time in tissue culture. This observation is noteworthy because mCpG-dependent silencing is thought to be the major mechanism for keeping genes on the inactive X chromosome silent. There is no direct evidence, however, that MeCP2 is involved in this process, as other MBD-containing proteins could play a role.
Histone H4, but not H3, is hyperacetylated in MeCP2-deficient cells
We hypothesized that MeCP2-truncating mutations may cause global histone hyperacetylation, because intact MeCP2 can form a complex with transcriptional co-repressors and HDACs that may influence nucleosome structure. An antibody was raised against a tetra-acetylated histone H4 N-terminal peptide. This rabbit antibody turned out to cross-react weakly with acetylated histone H3 (Fig. 2). On western blot (Fig. 3C), histone H4 (
10 kDa) was hyperacetylated in the female mutant cell lines 2 and 3, compared with control cell line 1 (see AcH4 bands). In contrast, acetylated histone H3 bands (AcH3) appeared to be of the same intensity in all three samples. Coomassie blue staining of gels run in parallel confirming equal protein loading (Fig. 3A). In Figure 3B and D, the male mutant sample was compared with the normal male control. AcH4 levels were increased, while AcH3 and Coomassie blue staining were identical. Quantitation of the signals by densitometry was used to calculate AcH4/AcH3, AcH4/H4 and AcH3/H3 ratios (Fig. 3E). Data from two independent experiments showed a consistent increase in acetylated H4 in the male and female mutant cell lines versus control samples, although the magnitudes differed. The results were further confirmed with a different antibody (see below).
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To further corroborate these results, the male mutant cells (803delG) were compared with the normal male control on western blots loaded at increasing protein concentrations (Fig. 4A). The densities of the histone H4 bands on the Coomassie blue stained gel run in parallel were recorded by densitometry. H4 acetylation levels increased corresponding to the amount of protein loaded (Fig. 4B). At all levels of concentration, the AcH4 bands, normalized for comparison between the two sets of samples, were distinctly stronger in the mutant sample than in the control, consistent with a 2- to 3-fold increase in global H4 acetylation (Fig. 4C).
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Hyperacetylation of histone H4 involves predominantly lysine residue 16
The N-terminal tail of histone H4 carries four different lysine residues, K5, K8, K12 and K16, that are subject to acetylation and deacetylation during chromatin modifications. The antibody used for the western blots shown in Figures 3C and D and 4B was directed against a histone H4 fragment acetylated at all four sites. To determine whether all sites contribute equally to the observed hyperacetylation in MeCP2 mutant cells, or whether specific K residues are preferentially involved, we carried out western analysis with antibodies against H4 acetylated at specific lysine residues. A series of western blots were prepared with the same five samples as shown in Figure 1. The levels of the chromatin-associated protein BRG1 were comparable in all samples (Fig. 5A), as were the levels of acetylated histone H3, detected with a commercial antibody specific for AcH3 (Fig. 5B). A commercial antibody specific for AcH4 confirmed the previously observed hyperacetylation of mutant samples 2, 3 and 4, compared with control samples 1 and 5 (Fig. 5C). With the antibodies against H4 acetylated at individual lysine residues, the most striking difference was seen for K16 (Fig. 5G). The pattern for K16 acetylation is very similar to that generated by the antibody to tetra-acetylated H4 in Figure 5C. Antibodies specific for the other acetylated lysines (K5, K8 and K12) revealed no discernible differences between the mutant and control samples. Data from up to four independent experiments and multiple scans were compiled as a comparison of the ratios of the mutant to control samples on the same western blots (Fig. 5H). Ratios plotted are a, 2/1, b, 3/1 and c, 4/5. Immunoprecipitation experiments have shown that antibodies to H4AcK16 recognize primarily the mono-acetylated isoform (29). We conclude that, for both truncating MECP2 mutations, the observed increase in H4 acetylation is predominantly due to acetylation of K16, reflecting an increase in histone H4 that is mono-acetylated at K16.
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DISCUSSION |
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The discovery of mutations in MECP2 as the cause of RTT came as a surprise (9). Although it provides a diagnostic test to confirm the clinical diagnosis in up to 80% of RTT patients, the processes by which absence of this ubiquitously expressed, abundant protein generates a neurological phenotype have yet to be unraveled. This task presents a major challenge. Although the current model predicts that MeCP2 is involved in controlling gene expression by affecting chromatin remodeling, no genes have yet been identified whose expression level is altered secondary to MeCP2 deficiency. Because of the previously demonstrated interaction of MeCP2 protein with a transcriptional repression complex that also contains HDACs (11), we hypothesized that endogenous truncating mutations which lack or disrupt the TRD may affect histone acetylation levels. By using a panel of antibodies against acetylated histones, we discovered that histone H4, but not H3, is hyperacetylated, specifically at lysine residue 16, in cell lines expressing only mutant MECP2 alleles.
To explain the selective hyperacetylation at H4K16, two possibilities have to be considered. First, the transcriptional repression complex that MeCP2 is associated with may contain a HDAC that is specific for H4AcK16. A repression complex lacking functional MeCP2 protein would not be able to recruit this HDAC, resulting in selective hyperacetylation of H4K16. The second, alternative, scenario is that MeCP2-mediated gene silencing specifically affects the gene for a HAT that preferentially targets H4K16 for acetylation. In MeCP2-deficient cells, the silencing of this putative HAT gene would be defective and could lead to hyperacetylation of H4K16. A HAT that acetylates chromatin specifically at histone H4 lysine 16 has indeed been discovered in Drosophila. It is identical to a previously known protein, called MOF, that is required for X chromosome dosage compensation. The mechanism of dosage compensation involves transcriptional activation of X-linked genes and is completely controlled by acetylation at H4K16 (30). Thus, acetylation of H4K16 could be a preferred mechanism for chromosome-wide epigenetic regulation of gene activity. Considering the histone code hypothesis (19,31), however, the cause and effect relationships between MeCP2-deficiency and increased H4K16 acetylation could be much more complex, involving several steps. For example, site-specific methylation or phosphorylation events on the N-terminal H4 tail could be altered as result of MeCP2 deficiency. Such altered modifications, if they occur, may change the histone tails’ ability to be recognized as a target for specific HDACs or HATs.
Regarding the location of the globally increased mono-acetylated H4AcK16 in MeCP2-deficient cells, many different sites in the genome could be affected. Immunofluorescence studies with antibodies against AcH4 demonstrated lack of acetylated histone H4 in centromere heterochromatin regions and the inactive X chromosome of mice and humans (32,33). Regional differences in AcH4 concentration along euchromatic chromosome arms were suggested by an R-banding-like pattern (34). An inverse correlation between anti-AcH4 and anti-MeCP2 staining has been reported for some regions. Whereas in mouse MeCP2 antibody binds most strongly to centromeric heterochromatin regions where the heavily methylated major satellite DNA is located (35), its distribution on human chromosomes appears to be more uniform (36).
Chromatin immunoprecipitation (ChIP) assays with antibodies to acetylated H4, however, did reveal associations of the H4AcK16 isoform with genomic regions or specific genes in human lymphoblasts (37). For example, c-myc, ß-globin, pro-insulin, growth hormone and 
-tubulin sequences were increased in the H4AcK16 fraction compared with the other H4AcK-specific fractions. There was no correlation, however, with the transcriptional status of the probed genes. This is not surprising, as histone acetylation is necessary but not sufficient for transcription. The H4AcK16 isoform was also relatively increased in chromatin associated with CpG islands and with genes located near heterochromatin regions. Most significantly, H4AcK16, as well as H4AcK8, were associated with the SINE/Alu family of short interstitial repeats which are enriched in R bands. Repetitive sequences located in constitutive heterochromatin were associated with under-acetylated H4 isoforms, whereas telomeric hexamer repeat sequences showed H4AcK16 levels similar to those in coding regions (37). To identify the sites of H4K16 hyperacetylation, and of possible target genes for MeCP2-dependent histone modification, immunofluorescence and ChIP studies with antibodies specific for H4AcK16 need to be performed on MeCP2-deficient cells.
The observed hyperacetylation of histone H4, but not of H3, in MeCP2-deficient cells may be functionally significant. Selective hyperacetylation of histone H4 has been reported to regulate gene activation of different loci under a variety of conditions. Changes in histone acetylation in response to inducible gene expression have been studied by ChIP assays of cell cultures, with and without prior hormone stimulation. In response to estrogen treatment, a transient significant increase (5- to 8-fold) in H4 acetylation levels was seen in the promoter region of target genes (38). H3 acetylation was also increased but to a lesser degree. Similarly, there was a preference for H4 hyperacetylation at the active FMR1 locus (39). In contrast, Bennett and Osborne (40), studying sterol-regulated gene expression of the low density lipoprotein receptor and HMG CoA-reductase genes, reported a selective increase of H3 acetylation in the sterol-induced cells at both promoters, whereas H4 acetylation levels remained similar to those of input DNA. After exposure of cells to the HDAC inhibitor trapoxin, transcription of the cell cycle regulator p21WAF1 was increased in association with an increase in the histone H3 acetylated fraction (41).
With respect to specific lysine residues, we observed global hyperacetylation only at H4K16. There was no difference between mutant and control cell lines for K8 and K12, whereas the results for K5 were less clear due to low signal and high background with the available antibody. This result may not be surprising if one considers that K16 is the most frequently acetylated lysine residue in all acetylated H4 isoforms, and the predominant one in the mono-acetylated form (29). Depending on cell type, K16 may be modified in up to 30% of the total H4 fraction. In contrast, acetylation at K5 is only found in tri- and tetra-acetylated isoforms that account for <2% of total histones. This may explain the weak signal with this antibody. There is evidence, however, that acetylation events may be interdependent. Acetylation of one lysine might require prior acetylation of another. Lysine acetylation acts in concert with other post-translational covalent histone modifications such as phosphorylation of serine residues or methylation, to establish the ‘histone code’ that is read by other protein complexes and results in modification of the chromatin structure of target genes (19,31).
Evidence for a functional role of H4K16 acetylation is provided by the existence of H4K16-specific HATs and HDACs. Aside from the MOF HAT in Drosophila, discussed above, lysine 16 is also a highly preferred acetylation site for GCN5, a yeast transcriptional adapter that has intrinsic HAT activity linking histone acetylation to gene activation and cell viability (42,43). Mutational studies in Saccharomyces cerevisiae indicated that H4K16 is critically important for efficient transcriptional silencing (44). SIR2 proteins which mediate transcriptional silencing in yeast and mammalian cells have an NAD-dependent HDAC activity that specifically targets H4K16 as well lysines 9 and 14 of H3. In vitro mutational studies showed that deacetylation of these specific lysine residues is essential for gene silencing (45). These reports suggest an important function of the acetylation status of H4K16 in regulating gene activity. Our findings of selective H4K16 acetylation provide a first step towards unraveling the pathogenesis of RTT.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell lines
Lymphoblastoid cell lines from two sisters with RTT of a previously described Brazilian family (8,10) were used for single cell cloning. After two washes in serum-free medium, cells were counted in a hemacytometer. Trypan blue (0.2%) exclusion was used as an indicator of viability. The cells were diluted to five viable cells/ml with conditioned medium, which was generated by filtration (0.2 µm filters) of medium collected after culturing lymphoblast cells for 2–3 days. By dispensing 200 µl of diluted cells, an average of one cell per well was placed in 96-well flat bottom tissue culture plates. About 5 weeks later, clusters of cells were transferred to 24-well tissue culture plates and then further expanded. A lymphoblastoid cell line from the male with congenital encephalopathy and a 803delG truncating MECP2 mutation, reported previously (10), was provided by Dr C. Schanen.
XCI assay
The XCI status of the parental cell lines and clonal cultures was established by studying methylation sensitive HpaII sites at a microsatellite within the AR locus on Xq11-q12, as described by Schanen et al. (6) and Allen et al. (28).
Expression of mutant and normal MECP2 alleles
For RT–PCR analyses, total RNA was extracted by using RNA STAT-60 (TEL-TEST), and poly(A)+ mRNA was purified with the Oligotex Direct mRNA kit (Qiagen). The Superscript Choice System was used for cDNA synthesis (GibcoBRL Life Technologies). Expression of the mutant R168X and normal MECP2 alleles in clonal female lymphoblastoid cell lines was assessed by RT–PCR using intron-spanning primers 5'-AGCCCGTGCAGCCATCAGCC-3' (in exon 3) and 5'-CTTCCCAGGACTTTTCTCCA-3' (in exon 4) (9). Since the 502CT (R168X) mutation generates a HphI site (10), the RT–PCR products were digested with HphI to distinguish the transcripts of the two alleles. Expression of the 803delG mutation in the male cells was detected by RT–PCR using exon 4 based primers, 5'-GGCAGGAAGCGAAAAGCTGAG-3' and 5'-TGAGTGGTGGTGATGGTGGTGG-3' (9), followed by NlaIV digestion of RT–PCR products (10).
Nuclear extract preparation and western blot with MeCP2 antibody
Cells (107) were washed twice with PBS and lysed with 10x volume lysis buffer (25 mM Hepes pH 7.8, 25 mM KCl, 5 mM MgCl2, 0.05 mM EDTA, 10% glycerol, 0.1% NP40, 0.1 mM PMSF, 1mM DTT) on ice for 5 min. Completeness of lysis was assessed by trypan blue (0.2%) staining. The nuclear pellet was washed with 10x volume lysis buffer to remove cytoplasmic contaminants and was resuspended in 10x volume nuclear extract buffer (10 mM Hepes pH 7.5, 200 mM NaCl, 1mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF), followed by incubation at 4°C for 15–30 min before centrifugation. The supernatant was used for western blot. Equal amounts of protein, as quantitated by using a protein assay kit (BioRad), were loaded onto 8% SDS–polyacrylamide gels. Proteins were transferred to a polyvinylidene difluoride membrane (Novex) and blocked with 5% non-fat milk in PBS at room temperature for 1 h. The membrane was incubated with rabbit polyclonal antibody against a well conserved peptide (PRPNREEPVDSRTP) at the C-terminal region of MeCP2 (catalog no. 07–013, Upstate Biotechnology) at room temperature for 2 h in PBS containing 0.1% Tween 20 and 2% non-fat milk. The membrane was then washed and probed with a horseradish peroxidase-conjugated secondary antibody in the same buffer. The bound antibody was detected with an ECL kit (Amersham).
Antibodies and western blots to detect histone acetylation
Antibody against acetylated histone H4 was raised by injecting KLH-conjugated tetra-acetylated N-terminal peptide (AGGAcKGGAcKGMGAcKVGAAcKRHSC) of histone H4 into rabbits. The antibody was affinity-purified by binding to peptide-conjugated Affi-gel 102 beads (Bio-Rad) and diluted 1:200. Rabbit polyclonal antibodies against acetylated histone H4 or H3, as well as against histone H4 acetylated at lysine residues 5, 8, 12 and 16 were purchased from Upstate Biotechnology. A rabbit anti-histone H4Ac16 antibody (catalog no. AHP417) was also purchased from Serotec (www.serotec.co.uk). All antibodies were diluted following the manufacturer’s instruction. The antibody against nuclear protein BRG1 was generously provided by G. Crabtree (Stanford University). SDS whole cell lysates were prepared by resuspending 107 cells in 200 µl of 2x SDS gel loading buffer (100 mM Tris–Cl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol). The samples were heated at 95°C for 5 min and sonicated gently to fragment the DNA. The cell lysates (10 µl each) were resolved by 18% SDS–PAGE and acetylated histones were detected by western blot as described above.
Densitometry and statistical analysis
The density of the signals on autoradiograms at different exposure times was measured by using the Quantity One and GS-710 Calibrated Imaging densitometer (Bio-Rad). Ratios of intensity were calculated for individual western blots by using internal controls or quantitation of stained H3 and H4 bands on parallel protein gels. Standard deviations were calculated using Microsoft Excel 98.
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ACKNOWLEDGEMENTS |
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We thank S. Naidu and C. Schanen for cell lines, G. Crabtree for the BRG antibody, T.H. Chi, V. Lemahieu and S. Fulmer-Smentek for stimulating discussion and K. Redman for administrative assistance. This work was supported by the Howard Hughes Medical Institute (U.F., S.S.J.L. and M.W.), an NIH research grant (U.F.), a Lynn Marie Chandler Postdoctoral Fellowship (M.W.) and intramural funds of the National Heart Lung and Blood Institute (K.Z.).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Genetics, Stanford University School of Medicine, Stanford CA 94305-5323, USA; Tel: +1 650 725 8089; Fax: +1 650 725 8112; Email: francke@cmgm.stanford.edu
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REFERENCES |
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