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Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome.

Brideau NJ, Coker H, Gendrel AV, Siebert CA, Bezstarosti K, Demmers J, Poot RA, Nesterova TB, Brockdorff N - Mol. Cell. Biol. (2015)

Bottom Line: We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms.A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome.Together, our results provide key insights into SMCHD1 function and target site selection.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

SMCHD1 conserved domains are required for chromatin loading. (A, top) Western blot showing subcellular fractionation of endogenous SMCHD1 and Smchd1−/− ES23 cells stably expressing WT SMCHD1-FLAG and mutant derivatives (Fig. 3B) in cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. Endogenous SMCHD1 from WT E14 ESCs is present in all fractions but predominantly in and approximately equally divided between the nuclear and chromatin-bound lanes. (Bottom) Histone H3 as a control for the chromatin-bound fraction and tubulin as a control for the cytoplasmic fraction. (B) Rabbit polyclonal anti-SMCHD1 antibody was tested by Western blotting against nuclear extracts from human HEK293T cells transfected with an SMCHD1-HA-monomeric GFP (mGFP) plasmid, wild-type mouse C127 fibroblasts, Smchd1−/− MEFs, Smchd1−/− ESCs stably expressing SMCHD1-FLAG (ES23+), and Smchd1−/− ESCs (ES23). Bands at the predicted molecular masses are visible in extracts containing SMCHD1 but not in extracts from Smchd1−/− cells. Asterisks indicate HA-mGFP-tagged protein. (C) Localization of SMCHD1 mutants in Smchd1−/− mouse embryonic fibroblasts. Cells were transfected with plasmids containing WT and derivative SMCHD1 constructs and stained with DAPI to mark DNA (blue), anti-K27me3 antibody for Xi (red), and anti-HA for SMCHD1 (green). The percentage of cells showing a focus for Xi is shown at the top right of each antibody panel, representing the means from 3 replicates (n > 50 cells). Bars, 5 μm.
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Figure 5: SMCHD1 conserved domains are required for chromatin loading. (A, top) Western blot showing subcellular fractionation of endogenous SMCHD1 and Smchd1−/− ES23 cells stably expressing WT SMCHD1-FLAG and mutant derivatives (Fig. 3B) in cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. Endogenous SMCHD1 from WT E14 ESCs is present in all fractions but predominantly in and approximately equally divided between the nuclear and chromatin-bound lanes. (Bottom) Histone H3 as a control for the chromatin-bound fraction and tubulin as a control for the cytoplasmic fraction. (B) Rabbit polyclonal anti-SMCHD1 antibody was tested by Western blotting against nuclear extracts from human HEK293T cells transfected with an SMCHD1-HA-monomeric GFP (mGFP) plasmid, wild-type mouse C127 fibroblasts, Smchd1−/− MEFs, Smchd1−/− ESCs stably expressing SMCHD1-FLAG (ES23+), and Smchd1−/− ESCs (ES23). Bands at the predicted molecular masses are visible in extracts containing SMCHD1 but not in extracts from Smchd1−/− cells. Asterisks indicate HA-mGFP-tagged protein. (C) Localization of SMCHD1 mutants in Smchd1−/− mouse embryonic fibroblasts. Cells were transfected with plasmids containing WT and derivative SMCHD1 constructs and stained with DAPI to mark DNA (blue), anti-K27me3 antibody for Xi (red), and anti-HA for SMCHD1 (green). The percentage of cells showing a focus for Xi is shown at the top right of each antibody panel, representing the means from 3 replicates (n > 50 cells). Bars, 5 μm.

Mentions: Antibodies to FLAG (catalog number F3165; Sigma), HA (clone 3F10; Roche), mCherry (catalog number ABE3523; Source Bioscience), green fluorescent protein (GFP) (catalog number ab290; Abcam), HP1α (catalog number MAB3584; Millipore), HP1γ (catalog number MAB3450; Millipore), histone H3 (catalog number ab1791; Abcam), H3K9me2 (catalog number 154-050; Diagenode), H3K9me3 (catalog number 05-1242; Millipore), H3K27me3 (catalog number 61017; Active Motif), and tubulin (catalog number 21445; Cell Signaling) were used in this study. Smchd1 rabbit polyclonal antibody was raised against a mixture of SMCHD1 fragments produced in bacteria (positions 1 to 385, 1197 to 1549, and 1615 to 1963), affinity purified, validated by Western blot analysis (see Fig. 5B), and used for experiments depicted in Fig. 5A, 7D, and 8. Smchd1 antibody (catalog number ab31865; Abcam) was used for experiments depicted in Fig. 1C and E.


Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome.

Brideau NJ, Coker H, Gendrel AV, Siebert CA, Bezstarosti K, Demmers J, Poot RA, Nesterova TB, Brockdorff N - Mol. Cell. Biol. (2015)

SMCHD1 conserved domains are required for chromatin loading. (A, top) Western blot showing subcellular fractionation of endogenous SMCHD1 and Smchd1−/− ES23 cells stably expressing WT SMCHD1-FLAG and mutant derivatives (Fig. 3B) in cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. Endogenous SMCHD1 from WT E14 ESCs is present in all fractions but predominantly in and approximately equally divided between the nuclear and chromatin-bound lanes. (Bottom) Histone H3 as a control for the chromatin-bound fraction and tubulin as a control for the cytoplasmic fraction. (B) Rabbit polyclonal anti-SMCHD1 antibody was tested by Western blotting against nuclear extracts from human HEK293T cells transfected with an SMCHD1-HA-monomeric GFP (mGFP) plasmid, wild-type mouse C127 fibroblasts, Smchd1−/− MEFs, Smchd1−/− ESCs stably expressing SMCHD1-FLAG (ES23+), and Smchd1−/− ESCs (ES23). Bands at the predicted molecular masses are visible in extracts containing SMCHD1 but not in extracts from Smchd1−/− cells. Asterisks indicate HA-mGFP-tagged protein. (C) Localization of SMCHD1 mutants in Smchd1−/− mouse embryonic fibroblasts. Cells were transfected with plasmids containing WT and derivative SMCHD1 constructs and stained with DAPI to mark DNA (blue), anti-K27me3 antibody for Xi (red), and anti-HA for SMCHD1 (green). The percentage of cells showing a focus for Xi is shown at the top right of each antibody panel, representing the means from 3 replicates (n > 50 cells). Bars, 5 μm.
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Figure 5: SMCHD1 conserved domains are required for chromatin loading. (A, top) Western blot showing subcellular fractionation of endogenous SMCHD1 and Smchd1−/− ES23 cells stably expressing WT SMCHD1-FLAG and mutant derivatives (Fig. 3B) in cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. Endogenous SMCHD1 from WT E14 ESCs is present in all fractions but predominantly in and approximately equally divided between the nuclear and chromatin-bound lanes. (Bottom) Histone H3 as a control for the chromatin-bound fraction and tubulin as a control for the cytoplasmic fraction. (B) Rabbit polyclonal anti-SMCHD1 antibody was tested by Western blotting against nuclear extracts from human HEK293T cells transfected with an SMCHD1-HA-monomeric GFP (mGFP) plasmid, wild-type mouse C127 fibroblasts, Smchd1−/− MEFs, Smchd1−/− ESCs stably expressing SMCHD1-FLAG (ES23+), and Smchd1−/− ESCs (ES23). Bands at the predicted molecular masses are visible in extracts containing SMCHD1 but not in extracts from Smchd1−/− cells. Asterisks indicate HA-mGFP-tagged protein. (C) Localization of SMCHD1 mutants in Smchd1−/− mouse embryonic fibroblasts. Cells were transfected with plasmids containing WT and derivative SMCHD1 constructs and stained with DAPI to mark DNA (blue), anti-K27me3 antibody for Xi (red), and anti-HA for SMCHD1 (green). The percentage of cells showing a focus for Xi is shown at the top right of each antibody panel, representing the means from 3 replicates (n > 50 cells). Bars, 5 μm.
Mentions: Antibodies to FLAG (catalog number F3165; Sigma), HA (clone 3F10; Roche), mCherry (catalog number ABE3523; Source Bioscience), green fluorescent protein (GFP) (catalog number ab290; Abcam), HP1α (catalog number MAB3584; Millipore), HP1γ (catalog number MAB3450; Millipore), histone H3 (catalog number ab1791; Abcam), H3K9me2 (catalog number 154-050; Diagenode), H3K9me3 (catalog number 05-1242; Millipore), H3K27me3 (catalog number 61017; Active Motif), and tubulin (catalog number 21445; Cell Signaling) were used in this study. Smchd1 rabbit polyclonal antibody was raised against a mixture of SMCHD1 fragments produced in bacteria (positions 1 to 385, 1197 to 1549, and 1615 to 1963), affinity purified, validated by Western blot analysis (see Fig. 5B), and used for experiments depicted in Fig. 5A, 7D, and 8. Smchd1 antibody (catalog number ab31865; Abcam) was used for experiments depicted in Fig. 1C and E.

Bottom Line: We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms.A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome.Together, our results provide key insights into SMCHD1 function and target site selection.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

No MeSH data available.


Related in: MedlinePlus