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. 2001 Nov 15;15(22):2940-4.
doi: 10.1101/gad.929101.

Lsh, a member of the SNF2 family, is required for genome-wide methylation

K Dennis  1 , T Fan, T Geiman, Q Yan, K Muegge
Affiliations

Lsh, a member of the SNF2 family, is required for genome-wide methylation

K Dennis et al. Genes Dev. .

Abstract

Methylation patterns of the mammalian genome are thought to be crucial for development. The precise mechanisms designating specific genomic loci for methylation are not known. Targeted deletion of Lsh results in perinatal lethality with a rather normal development. We report here, however, that Lsh(-/-) mice show substantial loss of methylation throughout the genome. The hypomethylated loci comprise repetitive elements and single copy genes. This suggests that global genomic methylation is not absolutely required for normal embryogenesis. Based on the similarity of Lsh to other SNF2 chromatin remodeling proteins, it suggests that alteration of chromatin affects global methylation patterns in mice.

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Figures

Figure 1
Figure 1
Hypomethylation of minor satellite sequences in Lsh−/− mice. (A) Southern analysis of genomic DNA derived at day 13.5 of gestation. DNA was digested with HpaII or MspI, blotted, and probed for minor satellite sequences using MR150. (B) Southern analysis of genomic DNA derived from newborn mice within 24 h after birth. Whole body comprises every tissue with the exception of the examined internal organs. DNA was digested with HpaII or MspI, blotted, and probed for minor satellite sequences using MR150.
Figure 2
Figure 2
Hypomethylation of repetitive sequences in Lsh−/− mice. Southern analysis for repetitive sequences. Genomic DNA derived from Lsh−/− or littermate controls was digested with MaeII and probed for major satellite sequences, or digested with HpaII and MspI (M) and probed for IAP, Sine B1, Line 1, or telomeric sequences.
Figure 3
Figure 3
Hypomethylation of single copy sequences in Lsh−/− mice. (A) Southern analysis of the β-Globin gene. Genomic DNA was derived from newborn mice or embryos at day 13.5 gestation. DNA was digested with BamHI with or without the methylation sensitive restriction enzyme HhaI, blotted, and probed for β-Globin. (B) Southern analysis of the Pgk-2 gene. (C) Southern analysis of the Pgk-1 gene. (D) Southern analysis of the H19 upstream imprinted region.
Figure 4
Figure 4
Global hypomethylation in Lsh−/− mice. (A) Genomic DNA from embryonal fibroblasts (MEF) or adult thymus from radiation chimeras (Geiman and Muegge 2000) was digested with the methylation-sensitive enzyme HpaII and the nonsensitive enzyme MspI, subjected to agarose gel electrophoresis, and visualized by ethidium bromide stain. (B) Methyl acceptance assay. Equal amounts of genomic DNA derived from day 13.5 embryonic body, embryonic liver, or from newborn brain were methylated in vitro by SssI CG methylase using radiolabeled S-adenosyl-methionine as donor. This approach allows determination of the amount of unmethylated CG sites in the genome and serves as an indirect measurement of genomic methylation levels. The amount of incorporated radiolabeled methyl groups on cytosines per microgram of DNA was measured in Lsh deleted samples and control littermates as described previously (Antoun et al. 2000). (C) Direct measurement of methyl-cytosine in genomic DNA. Equal amounts of genomic DNA derived from brain samples of Lsh−/− mice and littermate controls were digested with MspI, radiolabeled at the 5′-ends, and digested with nuclease P1 to generate 5′-deoxymononucleotides. Cytosine and methyl-cytosine were separated by thin layer chromatography (Cedar et al. 1979) and quantified using PhosphorImager analysis. The ratio of methyl-cytosine to total cytosine indicates the level of methylation at all CCGG sites. HpaII digests should not generate methyl-cytosine spots and serve as controls, indicating the specificity of the assay.
Figure 5
Figure 5
Expression of DNA methyltransferases and measurement of Mtase activity in Lsh−/− mice. (A) RT–PCR analysis. Total RNA of embryonic body (2 wild type, 1 heterozygote, and 3 knockout) derived from day 17.5 gestation was reverse transcribed and subjected to real-time PCR analysis for measurement of Dnmt1, Dnmt3a, or Dnmt3b or Gapdh transcripts as control. (CT) Cycle threshold, cycle number at which each PCR reaction reaches a predetermined fluorescence threshold, set within the linear range of all reactions. (B) Western analysis. Cellular extracts derived from fetal brain tissue were analyzed using specific antiserum against murine Dnmt1, Dnmt3a (Imgenex), Dnmt3b (Affinity Bioreagents), β-Actin (Sigma), or PCNA (Santa Cruz) as control. A similar result was obtained using lysates derived from embryonic body of day 17.5 gestation. (C) Mtase activity. Cellular extracts were prepared from indicated tissues and examined in vitro for their ability to transfer radio-labeled methyl-groups onto synthetic template poly[d(I–C)]·poly[d(I–C)] (Li et al. 1992). Embryonic bodies are from day 13.5 gestation.
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