Dunican DS et al. (2015), Lsh Is Essential for Maintaining Global DNA Met...

XB-ART-53349

Biomed Res Int 2015 Jan 01;2015:740637. doi: 10.1155/2015/740637.

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

Dunican DS , Pennings S , Meehan RR .

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Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides, a pattern which is maintained through cell division by the DNA methyltransferase Dnmt1. Dramatic methylation losses are observed in plant and mouse cells lacking Lsh (lymphoid specific helicase), predominantly at repetitive sequences and gene promoters. However, the mechanism by which Lsh contributes to the maintenance of DNA methylation is unknown. Here we show that DNA methylation is lost in Lsh depleted frog and fish embryos, both of which exhibit developmental delay. Additionally, we show that both Lsh and Dnmt1 are associated with chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association. Coimmunoprecipitation experiments reveal that Lsh and Dnmt1 are found in the same protein complex, and pulldowns show this interaction is direct. Our data indicate that Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1α-dependent manner. These data together (a) show that the role of Lsh in DNA methylation is conserved in plants, amphibian, fish, and mice and (b) support a model in which Lsh contributes to Dnmt1 binding to chromatin, explaining how its loss can potentially lead to perturbations in DNA methylation maintenance.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: dnmt1 emd hells tbl1x
GO keywords: DNA methylation [+]
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	Figure 1. Lsh is essential for both Xenopus laevis and Danio rerio development. (a–c) Xenopus laevis embryos were injected with xLMO or control morpholinos and allowed to develop. Each panel shows examples of morphant embryos and a control embryo (black arrows). xLMO is fluorescein labelled and successfully injected embryos can be visualised under UV light (c). Developmental stages are (a) 28, 37-38, 42, (b) 42–45, (c) 42–45. Scale bar = 1 mm. (d) In vitro inhibition of xLsh coupled transcription-translation (TNT) with xLMO. 35S-Methionine labelled xLsh protein was prepared by TNT in the presence or absence of xLMO and products separated by PAGE. xLsh production was inhibited by xLMO (compare left and middle lanes). Band on lower right is TNT luciferase protein. (e) Danio rerio embryos were injected with zLMO and allowed to develop to the midsomite stage (24 hpf). Severity of phenotype is dose-dependent (compare panels left to right). UV light showing successful microinjection of three doses of zLMO and severity of phenotype (top panel, lateral view). Brightfield view of three doses of zLMO (middle panel, lateral view). Two representative brightfield control morpholino injected embryos (lower panel, lateral view). Scale bar = 300 μm. (f) Southern blot analysis of genomic DNA isolated from control- and xLMO-injected tadpole embryos using a dispersed repeat xSatI probe. DNA was digested with either HpaII (methylation-sensitive) or MspI (methylation-insensitive HpaII isoschizomer), resolved and probed with radiolabelled xSatI. Digestion with HpaII indicates that xLMO DNA from tadpoles is more frequently cut as indicated by the low molecular weight banding pattern (black arrows) compared to control-injected genomic DNA. (g) Southern blot analysis of genomic DNA isolated from control- and zLMO-injected 24 hpf embryos using a Danio rerio Dana probe. A similar approach was taken as in (f). Compare the extent of HpaII digestion in lane 3 (control) and lane 4 (zLMO). Black bracket = wild type HpaII profile; dashed red bracket = zLMO HpaII profile. DNA sizes are indicated in kilobases to the left of each gel. (h) Upper: dot blot of Xenopus laevis genomic DNA probed with 5-methylcytosine antibody. Note the weaker binding of antibody to the xLMO DNA indicating global hypomethylation; lower: dot blot of Danio rerio genomic DNA probed with 5-methylcytosine antibody. Note the reduced binding of antibody to the zLMO DNA indicating global hypomethylation. (i) Summary of bisulfite sequencing of xSat in wild type and xLMO tadpole embryos. Vertical axis: % methylation; horizontal axis: each CpG in xSat amplicon.
	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 p53−/− MEF. White arrows indicate less frequent colocalisation with pericentric heterochromatin. n = 100. (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). n = 100. (g) Coexpression of Lsh and HP1α drives Lsh to heterochromatin. n = 80. (h-i) HP1α mutants (V21M-chromodomain and A129R-chromoshadow domain) do not redirect Lsh to heterochromatin. n = 90.
	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.
	Figure 4. Model for Lsh and Dnmt1 cooperation in silencing. (a) Model for Lsh:Dnmt1 mediated repression. In wild type cells, the H3K9trime mark acts as a ligand in HP1α recruitment to silent regions of the genome. Taking together our data and that of others, both Dnmt1 and Lsh can be associated with HP1α (perhaps requiring HDACs 1 and 2) thereby allowing the parallel docking of DNA methyltransferase and chromatin remodelling activities to silent loci. (b) In Lsh depleted cells (and knockout plants and animals), targeting of Dnmt1 is diminished leading to reduced DNA methylation maintenance and partial genomic hypomethylation. The accumulation of the activating H3K4me2 mark in Lsh−/− cells may be a downstream effect of DNA hypomethylation.
	Supp S1 . Comparison of xLSH protein sequence to non-Xenopus LSH homologues. A) Shown is a protein sequence alignment of LSH proteins from mammals to yeast. The numbers shown on each protein subdomain indicate the % identity of each LSH protein to the murine form. B) Phylogenetic trees were generated using ClustalW (www.ebi.ac.uk) of the LSH Snf-2 domain and the LSH helicase domain.
	Supp S2 . xLSH expression pattern during development A) Shown is temporal RT-PCR analysis of xLSH during embryonic development from unfertilised egg cells to fully developed tadpoles and the Xenopus kidney-derived cell-line A6. RT-PCR was also carried out for xGAPDH as a loading control. B) Shown is the spatial RNA in situ hybridisation pattern using an xLSH antisense RNA probe. Stages shown are: E=egg, bl=blastula, ga=gastrula, ne=neurula, tb=tailbud, td=tadpole, A6=A6 cell-line, -RT=-reverse transcriptase RT-PCR control. Both assays reveal xLSH to be semi-ubiquitous, indicating its importance during each stage 3 of development.
	Supp S3 . xLSH morphants are not apoptotic. a) Shown are early tadpole xLMO morphant embryos which show moderate Tunel-positive signals (indicating apoptosis) compared to a wildtype control of the same stage (c) which has no detectable Tunel signals. b) Shown are tadpole xLMO morphant embryos which show moderate Tunel-positive signals (indicating apoptosis) compared to a wildtype control of the same stage (d). Note in (d) the presence of many Tunel positive cells which imply cell lineages undergoing metamorphosis accompanied by cell death. Black arrows indicate Tunel-positive cells. All microinjections are in each blastomere of a 2-cell embryo; morpholino amount = 5-10ng.
	Supp. S4 Comparison of xLMO and control injected embryos. a) Shown is a graph which details the number of phenotypically normal embryos at each corresponding developmental stage. Control injected embryos are shown in blue and xLMO injected morphants are shown in pink. At stage 16 (neurulation) only 70% of morphants appear normal compared to >96% for the control siblings. Later in development (between stages 26 (tailbud) and 43 (tadpole)) the % of phenotypically normal morphant embryos decreases markedly to zero at stage 43. b) Shown are the percentages of normal embryos through development. Comparison of the xLMO bars indicates that after initial lethality at stage 16 morphant embryos do not die subsequently but suffer from developmental delay.
	Supp. S5 Comparison of zLMO, control morpholino and non-injected fish embryos. (a) Survival charts comparing zLMO, CMO and non-injected embryos between 24 and 48hr post-fertilisation. Phenotype charts comparing zLMO, CMO and non-injected embryos between 24 and 48hr post-fertilisation. (b) Graphical representation of the chart data in (a).
	Supp S6. Ethidium bromide staining, methylene blue staining of dot-blot DNA and coomassie gel of purified GST-fusion proteins used in GST-pulldown experiments. (a) Ethidium gels of digests from Figure 1f-g. (b) Methylene blue staining of Xenopus laevis wildtype and xLMO tadpole stage DNA over three dilutions as in Figure 1. (c) Asterisks indicate each GST-fusion protein which are detailed below the gel. Some fusions show strong induction in E. coli whereas others show weaker induction. Indeed, some fusions show more breakdown products than others. For the experiments in the main body of this paper (Fig. 2), care was taken to ensure that regardless of which GST-fusion was used that its concentration was not limiting.

	Supp S8 . xDnmt3a expression pattern during development. Shown is temporal RT-PCR analysis of xDnmt3aL1 during embryonic development from unfertilised egg cells to fully developed tadpoles and the Xenopus kidney-derived cell-line A6. Stages shown are: E=egg, bl=blastula, ga=gastrula, ne=neurula, tb=tailbud, td=tadpole, A6=A6 cell-line, -RT=-reverse transcriptase RT-PCR control

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Agoston, Retinoblastoma pathway dysregulation causes DNA methyltransferase 1 overexpression in cancer via MAD2-mediated inhibition of the anaphase-promoting complex. 2007, Pubmed