Lsh Is Essential for Maintaining Global DNA Methylation Levels in Amphibia and Fish and Interacts Directly with Dnmt1

Figure 2

Lsh and Dnmt1 proteins interact in vitro and in vivo and Lsh is predominantly excluded from pericentric heterochromatin. (a) Cartoon of Lsh and Dnmt1 GST-fusions used. Individual fusions are indicated by numbering under each protein. (b) Direct interaction between Lsh and Dnmt1. Top: mLsh GST-fusions 1–208 and 560–822 pulldown radiolabelled full-length mDnmt1. Bottom: mDnmt1 GST pulldown radiolabelled full-length mLsh. All assays performed in the presence of 50 μg/mL ethidium bromide. (c) Full-length tagged Dnmt1 and Lsh can interact in vivo in cultured cells. Tagged proteins (GFP-xDnmt1 and T7-xLsh) were transfected into 293T cells and immunoprecipitated under high salt conditions (250 mM NaCl). Both proteins coimmunoprecipitate reciprocally (see IP lanes, right of each panel). (d) Endogenous immunoprecipitation of human Lsh and Dnmt1 in SW620 cells. (e) Lsh is predominantly nuclear diffuse. Expression of tagged (cherry red) mLsh in MEF. White arrows indicate less frequent colocalisation with pericentric heterochromatin. . (f) Expression of previously published [29] GFP-tagged mLsh is nuclear diffuse; in contrast, expression of GFP-tagged HP1α overlaps with pericentric heterochromatin foci (white arrows). . (g) Coexpression of Lsh and HP1α drives Lsh to heterochromatin. . (h-i) HP1α mutants (V21M-chromodomain and A129R-chromoshadow domain) do not redirect Lsh to heterochromatin. .

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