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The Human Proteins MBD5 and MBD6 Associate with Heterochromatin but They Do Not Bind Methylated DNA

Laget, Sophie, Joulie, Michael, Le Masson, Florent, Sasai, Nobuhiro, Christians, Elisabeth, Pradhan, Sriharsa, Roberts, Richard J., Defossez, Pierre-Antoine·2010·PLoS ONE
DOI 10.1371/journal.pone.0011982PMC PMC2917364
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Figure 1 Organization of the human MBD5 and MBD6 genes and proteins. A- The coding exons of human MBD5 (top) and MBD6 (bottom). Translation of exons 6–15 of MBD5 yields protein isoform 1; translation of exons 6–9 with retention of the following intron yields protein isoform 2. B- Organization of the human MBD5 and MBD6 proteins. Amino-acid numbers are indicated. P rich: proline-rich segment; PWWP: PWWP domain; NLS: putative nuclear localization signal. The amino-acids that are unique to MBD5 isoform 2 are depicted in blue. C- Sequence comparison of all known human MBD domains. Periods, colons, and stars indicate increasing conservation of a given residue. The arrow shows the residue we mutated in MBD5 for further experiments. The secondary structure of MeCP2 is shown at the top. ß: Beta-sheet; α: alpha-helix; L: loop.
Figure 1 Organization of the human MBD5 and MBD6 genes and proteins. A- The coding exons of human MBD5 (top) and MBD6 (bottom). Translation of exons 6–15 of MBD5 yields protein isoform 1; translation of exons 6–9 with retention of the following intron yields protein isoform 2. B- Organization of the human MBD5 and MBD6 proteins. Amino-acid numbers are indicated. P rich: proline-rich segment; PWWP: PWWP domain; NLS: putative nuclear localization signal. The amino-acids that are unique to MBD5 isoform 2 are depicted in blue. C- Sequence comparison of all known human MBD domains. Periods, colons, and stars indicate increasing conservation of a given residue. The arrow shows the residue we mutated in MBD5 for further experiments. The secondary structure of MeCP2 is shown at the top. ß: Beta-sheet; α: alpha-helix; L: loop.CC BY · PubMed Central
Figure 2 Detection of MBD isoforms 1 and 2 in cell extracts. Human MCF7 cells in culture were transfected with the indicated expression plasmids. Total cell extracts were probed, in parallel, with anti-HA antibodies to detect exogenous proteins, and with anti-MBD5 antibodies to detect both endogenous and exogenous proteins. Top: long exposure of the high-molecular weight region demonstrates the existence of Isoform 1. Middle: Isoform 2 is detected as a 110-kDa-migrating species. Bottom: detection of Tubulin proves equal loading between samples. The arrows indicate the two MBD5 isoforms; position of migration markers, with their indicative weight in kDa, is indicated.
Figure 2 Detection of MBD isoforms 1 and 2 in cell extracts. Human MCF7 cells in culture were transfected with the indicated expression plasmids. Total cell extracts were probed, in parallel, with anti-HA antibodies to detect exogenous proteins, and with anti-MBD5 antibodies to detect both endogenous and exogenous proteins. Top: long exposure of the high-molecular weight region demonstrates the existence of Isoform 1. Middle: Isoform 2 is detected as a 110-kDa-migrating species. Bottom: detection of Tubulin proves equal loading between samples. The arrows indicate the two MBD5 isoforms; position of migration markers, with their indicative weight in kDa, is indicated.CC BY · PubMed Central
Figure 3 MBD5 and MBD6 are differentially expressed in mouse tissues. A-MBD5 Isoform 1 is expressed at highest levels in the brain, whereas isoform 2 is most expressed in oocytes. B- MBD6 is expressed at highest levels in the testis. Expression levels were measured by quantitative RT-PCR, with normalization to the RPS16 gene.
Figure 3 MBD5 and MBD6 are differentially expressed in mouse tissues. A-MBD5 Isoform 1 is expressed at highest levels in the brain, whereas isoform 2 is most expressed in oocytes. B- MBD6 is expressed at highest levels in the testis. Expression levels were measured by quantitative RT-PCR, with normalization to the RPS16 gene.CC BY · PubMed Central
Figure 4 MBD5 and MBD6 can colocalize with methylated regions in mouse nuclei. The indicated proteins were transfected into mouse 3T3 cells. The distribution of the various proteins in fixed cells was recorded, and representative images are provided. When different types of localization occurred, their approximate proportion is indicated. A-MBD5 isoform 1 always colocalizes with the chromocenters, DAPI-dense regions that harbor hypermethylated heterochromatin; mutating either MBD or PWWP domain changes this distribution to a diffuse nuclear pattern. Isoform 2 is also incapable of chromocenter localization. B- MBD6 colocalizes with chromocenters in 25% of the observed cells. Upon deletion of the MBD, the pattern becomes diffuse in all cells.
Figure 4 MBD5 and MBD6 can colocalize with methylated regions in mouse nuclei. The indicated proteins were transfected into mouse 3T3 cells. The distribution of the various proteins in fixed cells was recorded, and representative images are provided. When different types of localization occurred, their approximate proportion is indicated. A-MBD5 isoform 1 always colocalizes with the chromocenters, DAPI-dense regions that harbor hypermethylated heterochromatin; mutating either MBD or PWWP domain changes this distribution to a diffuse nuclear pattern. Isoform 2 is also incapable of chromocenter localization. B- MBD6 colocalizes with chromocenters in 25% of the observed cells. Upon deletion of the MBD, the pattern becomes diffuse in all cells.CC BY · PubMed Central
Figure 5 MBD5 and MBD6 can be recruited to chromocenters in Dnmt1−/− cells. The various GFP fusions were transfected into mouse fibroblasts of the indicated genotype. A -ZBTB4, MBD5, and MBD6, are recruited to the chromocenters of Dnmt1+/+ cells. B- MBD5 and MBD6 can be recruited to the demethylated chromocenters of Dnmt1−/− cells; ZBTB4 does not overlap with the chromocenters in the same cells.
Figure 5 MBD5 and MBD6 can be recruited to chromocenters in Dnmt1−/− cells. The various GFP fusions were transfected into mouse fibroblasts of the indicated genotype. A -ZBTB4, MBD5, and MBD6, are recruited to the chromocenters of Dnmt1+/+ cells. B- MBD5 and MBD6 can be recruited to the demethylated chromocenters of Dnmt1−/− cells; ZBTB4 does not overlap with the chromocenters in the same cells.CC BY · PubMed Central
Figure 6 The MBD domain of MBD5 and MBD6 does not bind methylated DNA in vitro. A- The MeCP2, MBD5 and MBD6 MBDs, tagged with 6×His, were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. Apparent weights of migration markers, in kDa, are indicated. B-Gel retardation assay with probe SL1 and the proteins of panel A. In this panel and the following, the probe is depicted as a line; open circles represent an unmethylated CpG, and filled circles a methylated CpG. In panels 2B–2D, the dash indicates the probe-only lane (no protein added). “MeCP2” : MBD domain of MeCP2, tagged with 6×His; same abbreviation for MBD5 and MBD6. C-Gel retardation assay with probe SL2 and the proteins of panel A. D-Gel retardation assay with probe SL3 and the proteins of panel A. E- The MeCP2, MBD5 and MBD6 MBDs, tagged with Maltose-Binding Protein (MBP), were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. F-Gel retardation assay with probe SL3, and the proteins of panel E. “MeCP2” : MBD domain of MeCP2 tagged with MBP; same abbreviation for MBD5 and MBD6. “MBP” : Maltose-Binding Protein only lane. G- The MBDs of MBD5 and MBD6, tagged with GST, were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. A region of MBD5 larger than just the MBD was included in this experiment, explaining the higher molecular weight. H-Gel retardation assay with probe SL4. “ZBTB4”: Zinc fingers of ZBTB4 fused to GST; “MBD5” : MBD domain of MBD5 fused to GST, same abbreviation for MBD6. “GST”: GST-only lane. I-Gel retardation assay with probe SL5 and the proteins used in panel H. Probe SL5 contains 6 randomized positions (“N”), on either side of the CpG. Legend as in panel H.
Figure 6 The MBD domain of MBD5 and MBD6 does not bind methylated DNA in vitro. A- The MeCP2, MBD5 and MBD6 MBDs, tagged with 6×His, were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. Apparent weights of migration markers, in kDa, are indicated. B-Gel retardation assay with probe SL1 and the proteins of panel A. In this panel and the following, the probe is depicted as a line; open circles represent an unmethylated CpG, and filled circles a methylated CpG. In panels 2B–2D, the dash indicates the probe-only lane (no protein added). “MeCP2” : MBD domain of MeCP2, tagged with 6×His; same abbreviation for MBD5 and MBD6. C-Gel retardation assay with probe SL2 and the proteins of panel A. D-Gel retardation assay with probe SL3 and the proteins of panel A. E- The MeCP2, MBD5 and MBD6 MBDs, tagged with Maltose-Binding Protein (MBP), were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. F-Gel retardation assay with probe SL3, and the proteins of panel E. “MeCP2” : MBD domain of MeCP2 tagged with MBP; same abbreviation for MBD5 and MBD6. “MBP” : Maltose-Binding Protein only lane. G- The MBDs of MBD5 and MBD6, tagged with GST, were expressed in bacteria, purified, and examined on a Coomassie-stained SDS-PAGE gel. A region of MBD5 larger than just the MBD was included in this experiment, explaining the higher molecular weight. H-Gel retardation assay with probe SL4. “ZBTB4”: Zinc fingers of ZBTB4 fused to GST; “MBD5” : MBD domain of MBD5 fused to GST, same abbreviation for MBD6. “GST”: GST-only lane. I-Gel retardation assay with probe SL5 and the proteins used in panel H. Probe SL5 contains 6 randomized positions (“N”), on either side of the CpG. Legend as in panel H.CC BY · PubMed Central

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Metadata

Authors
Laget, S., Joulie, M., Le Masson, F., Sasai, N., Christians, E., Pradhan, S., Roberts, R. J., & Defossez, P.-A.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2010-08-06
Volume
5
Issue
8
Pages
e11982
ISSN
1932-6203
DOI
10.1371/journal.pone.0011982
PMC
PMC2917364
Cited by
110
References
5
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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