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First published online 11 March 2008
doi: 10.1242/jcs.016865

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Analysis of protein domains and Rett syndrome mutations indicate that multiple regions influence chromatin-binding dynamics of the chromatin-associated protein MECP2 in vivo

¹ Nemours Biomedical Research, Alfred I duPont Hospital for Children, Wilmington, DE 19803, USA
² Department of Computer and Information Sciences, University of Delaware, Newark, DE 19716, USA
³ Department of Biological Science, Hokkaido Institute of Public Health, N-19 W-12, Kita-ku, Sapporo 060-0819, Japan
⁴ Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
⁵ Department of Medical Microbiology and Immunology, University of California, Davis, CA 95616, USA

View larger version (44K): [in this window] [in a new window]   Fig. 1. MECP2e1 and MECP2e2 colocalize in pericentromeric heterochromatin along with other heterochromatin marker proteins. (A) Balb/c 3T3 cells expressing EGFP-tagged MECP2e1 and MECP2e2 were stained with the DNA stain DAPI and imaged by epifluorescence microscopy. Bar, 5 µm. (B) Balb/c 3T3 cells were co-transfected with MECP2e2 tagged with ECFP and MECP2e1 tagged with EGFP. Cells were induced with 100 µM Zn2+ and imaged by confocal microscopy applying the online fingerprinting mode to separate the overlapping spectra. Bar, 1 µm. (C) Cells expressing EGFP-tagged MECP2e2 were immunostained with heterochromatin protein 1 (HP1) and histone H3 trimethylated at lysine 9. Bar, 5 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 2. MECP2e1 and MECP2e2 are mobile in vivo and demonstrate identical kinetics. (A) FRAP was performed on EGFP-tagged MECP2e1 and MECP2e2. Individual pericentromeric heterochromatin foci were photobleached and the recovery was monitored over time. The intensity in the photobleached region was normalized to total nuclear intensity and plotted as a function of time. The recovery curves indicate rapid binding kinetics of both protein isoforms and nearly complete recovery of fluorescence within the bleached area. (B) Early recovery kinetics for the two protein isoforms were examined by performing FRAP experiments at a high scan speed of 323 mseconds. The rapid recovery kinetics of MECP2e2 in euchromatin are also shown. HC, heterochromatin; EU, euchromatin. (C) The upper panels demonstrate a bleaching sequence of a heterochromatic focus in a MECP2e2-expressing cell. The lower panels show a similar sequence in a euchromatic area of the nucleus. Bar, 5 µm. (D) The t50 values were determined by fitting the recovery curves. The t50 values of the two isoforms in heterochromatin are not statistically significant (MECP2e1=20.8±5.6 and MECP2e2=21.3±5.4). By contrast, the mobility of MECP2e2 in euchromatin is extremely rapid (t50=0.125±0.003; *P<0.0001). The number of nuclei photobleached is shown within each bar.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Treatment with the DNA demethylating agent 5-Aza-2-deoxycytidine does not significantly alter MECP2e2 dynamics. (A) Balb/c 3T3 cells were treated with 1 µM 5-Aza-2-deoxycytidine (5-Aza-dC) for 5 days then transiently transfected with the human MECP2e2-GFP construct driven by the CMV promoter. 48 hours post-transfection, cells were photobleached. The recovery profiles of 5-Aza-dC-treated and untreated cells in heterochromatic regions are shown. The slight leftwards shift in the recovery curve was noted. (B) Recovery kinetics of 5-aza-dC treated and vehicle (DMSO) control in euchromatic regions are shown and indicate no changes in mobility associated with drug treatment. (C) Although there was a slight shift in the recovery curve in heterochromatin, the differences in the t50 values following 5-Aza-dC treatment were not statistically significant in either heterochromatin and euchromatin. The t50 values of vehicle and 5-Aza-dC treated cells in heterochromatin are 20.9±10.2 and 13.3±6.1, respectively (P=0.0798). In euchromatin, the t50 values of vehicle and 5-Aza-dC-treated cells are 0.13±0.01 and 0.13±0.008, respectively (P=0.7561). The number of nuclei photobleached in each group is shown within the bar.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Expression constructs of MECP2 and their cellular localization. (A) Schematic showing the expression constructs of MECP2 that were used in the domain-deletion studies. Deleted residues based on murine MECP2e2 are shown in parentheses. The locations of NLS are shown as red boxes. (B) Cells expressing EGFP-tagged WT and mutant proteins were stained with DAPI and imaged by epifluorescence microscopy. The cellular distribution of the proteins is indicated (N, nuclear; HC, heterochromatin; C, cytoplasmic). (C) Deletion of the MBD results in the mislocalization of MECP2 to the nucleolus. Stable cell lines expressing EGFP-tagged WT and MBD were immunostained with the nucleolar marker nucleolin and counterstained with DAPI. (D) Cells expressing the TRD mutant showing both cytoplasmic and nuclear localization. Bars, 5 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Deletion of the N- or C-terminus of MECP2 does not have any significant impact on its mobility. (A) Cells expressing EGFP-tagged MECP2e2 (WT), N and C were photobleached and their recovery curves were plotted, revealing essentially no differences in binding kinetics for the deleted proteins. (B) Curve fitting was used to determine t50 values±s.d. for each of the protein forms (21.3±5.4, 17.6±9.3 and 23.9±7.8 for WT, N and C, respectively). The t50 values of N and C are not significantly different from WT (P>0.05). The number of nuclei photobleached in each group is shown within the bars.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Deletion of the MBD, interdomain region and the TRD impact mobility of MECP2. (A) Recovery kinetics of the domain-deleted mutant forms indicate that that the MBD, ID and TRD contribute to the mobility. (B) The t50 values for the domain-deleted proteins indicate increased mobility for the protein within heterochromatin domains for the ID and TRD proteins compared with WT. The t50 for the MBD in nucleoplasm was intermediate between the value for euchromatin and heterochromatin for the WT protein (WT=21.3±5.4, MBD=1.3±0.4, ID=5.3±2.1 and TRD=7.9±2.5). Asterisks represent t50 values that are significantly different from heterochromatin in WT (P<0.0001). Number of nuclei for each group is shown within each bar.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Common RTT mutations significantly impact mobility of MECP2. (A) Recovery profiles of MECP2e2 (WT) and common RTT mutations of MECP2 found in the patient population. Balbc/3T3 cells were transiently transfected with CMV driven expression constructs encoding N-terminus EGFP-tagged MECP2, including common RTT mutations. Photobleaching was performed in regions of nucleoplasm (R106W, F155S, T158M) or heterochromatin (R133C, R168X). (B) The t50 values of the WT and RTT mutants (WT=23.4±8.8, R106W=1.4±0.42, R133C=12.4±4.5, F155S=1.5±0.5, T158M=1.4±0.3 and R168X=1.17±0.5). The sample size for each group is given within the bars. Asterisks represent significant different values compared with the WT protein in heterochromatin (P<0.0001).

View larger version (18K): [in this window] [in a new window]   Fig. 8. The t50 value of R255X does not differ significantly from WT protein. The binding kinetics for the R255X mutant protein are not significantly different than WT. A two-tailed t-test does not reveal a statistically significant difference in the t50 values between the two groups (P=0.0608). The number of nuclei is shown within each bar.