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

Figure 3

Lsh is associated with chromatin and is required for Dnmt1-chromatin association. (a) MNase treatment of 293T nuclei indicates that endogenous Lsh and Dnmt1 are chromatin bound (see untreated lanes). (b) Endogenous Lsh is associated with soluble chromatin. Sucrose gradient sedimentation was used to fractionate 3T3 soluble chromatin and both protein and genomic DNA were isolated from each fraction. Fractionation of chromatin was validated by DNA gel electrophoresis of all gradient fractions. Western blotting of fractions shows that mLsh (free) is enriched at the top of the gradient (open chromatin) and also cosediments with bulk chromatin (chromatin bound) in the middle and end of the gradient (compact chromatin). (c) siRNAs against human Lsh were tested in knockdown experiments in 293T cells and siLsh#3 gives ~70% knockdown. (d) Lsh is required for the Dnmt1-chromatin association. Comparison of wild type and siRNA treated 293T cells by MNase treatment of nuclei shows that Dnmt1:chromatin association is decreased in knockdown cells (comparison of amounts of Dnmt1 released into the supernatant show higher levels released in knockdown cells). Densitometry of the western blots shows that Dnmt1 is enriched in the chromatin bound fraction (left panel); knockdown of Lsh shifts Dnmt1 into the unbound fraction. Emerin was used as a control for a protein which is unaffected by MNase treatment.

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