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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: Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins.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.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus

SMCHD1 does not predominantly colocalize with HP1 proteins at pericentric regions. (A) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and Lrif1- (clone G5) ES23+ cells into cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. (B) SMCHD1 staining in WT J1 XY ESCs. Pericentric heterochromatin domains are visualized with HP1α and are also visible in DNA (DAPI) panels. The percentage of cells that contain either broad nuclear staining (top) or pericentric staining (bottom) are listed at the top right corner of the SMCHD1 panels. Scoring data are the means from 3 replicates (n > 150 cells). (C and D) SMCHD1 staining in WT MEFs. (C) Staining for HP1α is enriched at pericentric heterochromatin domains but not Xi, which is enriched for SMCHD1. (D) HP1γ enrichment at pericentric foci and also throughout the chromatin arms is seen in a broad staining pattern more similar to that of SMCHD1. Note the two Xi foci in panel D. Bars, 5 μm. (E) Western blotting for HP1γ on nuclear extracts from WT and HP1γ- (clone H5) ES23+ cells. Histone H3 is shown as a control. (F) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and HP1γ- (clone H5) ES23+ cells.
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Figure 7: SMCHD1 does not predominantly colocalize with HP1 proteins at pericentric regions. (A) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and Lrif1- (clone G5) ES23+ cells into cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. (B) SMCHD1 staining in WT J1 XY ESCs. Pericentric heterochromatin domains are visualized with HP1α and are also visible in DNA (DAPI) panels. The percentage of cells that contain either broad nuclear staining (top) or pericentric staining (bottom) are listed at the top right corner of the SMCHD1 panels. Scoring data are the means from 3 replicates (n > 150 cells). (C and D) SMCHD1 staining in WT MEFs. (C) Staining for HP1α is enriched at pericentric heterochromatin domains but not Xi, which is enriched for SMCHD1. (D) HP1γ enrichment at pericentric foci and also throughout the chromatin arms is seen in a broad staining pattern more similar to that of SMCHD1. Note the two Xi foci in panel D. Bars, 5 μm. (E) Western blotting for HP1γ on nuclear extracts from WT and HP1γ- (clone H5) ES23+ cells. Histone H3 is shown as a control. (F) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and HP1γ- (clone H5) ES23+ cells.

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 does not predominantly colocalize with HP1 proteins at pericentric regions. (A) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and Lrif1- (clone G5) ES23+ cells into cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. (B) SMCHD1 staining in WT J1 XY ESCs. Pericentric heterochromatin domains are visualized with HP1α and are also visible in DNA (DAPI) panels. The percentage of cells that contain either broad nuclear staining (top) or pericentric staining (bottom) are listed at the top right corner of the SMCHD1 panels. Scoring data are the means from 3 replicates (n > 150 cells). (C and D) SMCHD1 staining in WT MEFs. (C) Staining for HP1α is enriched at pericentric heterochromatin domains but not Xi, which is enriched for SMCHD1. (D) HP1γ enrichment at pericentric foci and also throughout the chromatin arms is seen in a broad staining pattern more similar to that of SMCHD1. Note the two Xi foci in panel D. Bars, 5 μm. (E) Western blotting for HP1γ on nuclear extracts from WT and HP1γ- (clone H5) ES23+ cells. Histone H3 is shown as a control. (F) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and HP1γ- (clone H5) ES23+ cells.
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Figure 7: SMCHD1 does not predominantly colocalize with HP1 proteins at pericentric regions. (A) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and Lrif1- (clone G5) ES23+ cells into cytoplasmic (Cy), soluble nuclear (N), and chromatin-bound (Ch) fractions. (B) SMCHD1 staining in WT J1 XY ESCs. Pericentric heterochromatin domains are visualized with HP1α and are also visible in DNA (DAPI) panels. The percentage of cells that contain either broad nuclear staining (top) or pericentric staining (bottom) are listed at the top right corner of the SMCHD1 panels. Scoring data are the means from 3 replicates (n > 150 cells). (C and D) SMCHD1 staining in WT MEFs. (C) Staining for HP1α is enriched at pericentric heterochromatin domains but not Xi, which is enriched for SMCHD1. (D) HP1γ enrichment at pericentric foci and also throughout the chromatin arms is seen in a broad staining pattern more similar to that of SMCHD1. Note the two Xi foci in panel D. Bars, 5 μm. (E) Western blotting for HP1γ on nuclear extracts from WT and HP1γ- (clone H5) ES23+ cells. Histone H3 is shown as a control. (F) Western blot showing the SMCHD1-FLAG distribution following subcellular fractionation of WT and HP1γ- (clone H5) ES23+ cells.
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: Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins.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.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus