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First published online 27 February 2008
doi: 10.1242/dev.016402

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xDnmt1 regulates transcriptional silencing in pre-MBT Xenopus embryos independently of its catalytic function

¹ Human Genetics Unit, MRC, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.
² Genes and Development group, School of Biomedical Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.
³ Institute of Gene Biology, Russian Academy of Sciences, Vavilova 34/5, Moscow, 119334, Russian Federation.

View larger version (73K): [in this window] [in a new window]   Fig. 1. xDMO embryos have reduced xDnmt1 levels, are abnormal and mis-express genes. (A) Top panel: in vitro inhibition of xDnmt1 translation (black arrowhead) using xDMO (compare lanes 2 and 3). Bottom panel: in vivo inhibition of xDnmt1 translation in pre-MBT (stage 7-8) embryos (compare wild-type and xDMO extracts). Tubulin is used as a loading control. (B) Left panel: phenotypes of stage 15 embryos. Morphant xDMO embryos exhibit apoptotic lesions (arrowheads and enlargement) and lack neural folds (black arrow) compared with control stage 15 embryos. xDMO embryos contain fluorescein, unlike the control embryo (compare arrowed embryos). Right panel: comparison of percentage (n=100) of successfully neurulating embryos for wild type and xDMO. (C) xDMO embryos mis-express a range of transcripts. Wild-type and xDMO RNA was assayed by RT-PCR over a 10-fold dilution range (0.1, 0.3 and 1 µl cDNA for each sample indicated by the black triangles). H4 is a loading control. (D) In situ analysis reveals ectopic expression of the indicated xDMO targets throughout the animal pole (compare wild-type and xDMO panels). The maternally expressed gene xOct60 is not mis-expressed. Scale bars: 1 mm in B,D. Animal pole views are shown.

View larger version (36K): [in this window] [in a new window]   Fig. 2. No changes in DNA methylation at repeat or single copy sequences in xDMO morphants. (A) xDMO DNA is heavily methylated at xSatI HpaII sites (compare lanes 2 and 4). HpaII is a methyl-sensitive restriction enzyme and MspI is the methylation insensitive (CCGG) counterpart (lane 5). Right panel: HindIII was used to generate the 750 bp xSatI monomer (black arrow); double digestion with HindIII and HpaII showed no difference in monomer methylation in the wild-type and xDMO samples (lanes 2-3). (B) Bisulphite sequencing (clones n=40) shows no significant difference in CpG methylation between wild-type and xDMO genomes at xSatI sequences. Boxed numbers are percentage CpG methylation; black circles indicate CpG distribution in xSatI. (C) CpG distribution in cloned promoters of xOct91 and xCycD1. Blue bars, CpG; black arrows, transcription start sites; red bars, regions sequenced. (D) Bisulfite analysis (sequences n=40, ten representative clones are shown) was used to determine the methylation status of xOct91 (left) and xCycD1 (right) promoters and upstream regions. Numbers above each CpG indicate genomic position relative to transcription start. Filled circles, methylated CpGs; empty circles, non-methylated CpGs. (E) Immunoblot analysis of wild-type and xDMO histones shows no significant change in various histone modification marks between histone WT and xDMO extracts at stages 8 and 15. Histone modifications are low to absent at stage 8 and accrue by stage 15. Black dots indicate non-specific bands.

View larger version (57K): [in this window] [in a new window]   Fig. 3. A catalytically inactive form of human DNMT1 can restore repression and rescue xDMO morphants. (A) Functional domains present in human DNMT1 and an inactivating point mutation of hDNMT1 (hDNMT1C1226Y) (Jair et al., 2006). (B) Catalytic function of human DNMT1 is not required to rescue xOct25 and xBF2 gene repression. Compare rescue in situ intensities (iii and iv) with signals in control (i) and xDMO morphants (ii). Animal pole views are shown. (C) hDNMT1WT and hDNMT1C1226Y restore normal Xenopus neurulation with comparable efficiency. Experiment 1 (n=100) is in red and experiment 2 (n=60) is in blue. Compare number of normally neurulating embryos in xDMO only (20%) with rescued injected embryos (>40%). (D) Rescued embryos are similar in phenotype to wild-type siblings. Neurulating embryos are shown. There are apoptotic cells and open blastopores (white arrowheads) in xDMO embryos (bottom left). Such lesions are absent in rescued embryos (right panels), which are similar to control injected embryos (top left) (black arrows indicate neural folds). (E) Phenotypes of late stage (stage 27) embryos. Only 2% (i.e. 98% of embryos fail) of xDMO morphants develop to late tadpole stage (stage 27) compared with 35% and 30% for wild-type (hDNMT1WT) and mutant (hDNMT1C1226Y) rescued embryos, which develop normally. Scale bars: 1 mm.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Dnmt1 represses activation of non-methylated transgenes in vivo and xDnmt1 localises to non-methylated target genes. (A) N-terminal xDnmt1 fusions bind dsDNA oligos. The domain structure of xDnmt1 (top). xDnmt1-Gst fusions domains G1-G3 (black bars). CpGpos oligonucleotides were used in pull-down assays with the three Gst fusions and Gst only. All three constructs bound CpGpos compared with the Gst protein (G) lane. (B) In vivo assays to test the repression activities of xDnmt1, hDNMT1WT and hDNMT1C1226Y. Exogenous xSp1 was used to activate the Sp1-Luc reporter in the presence of increasing amounts of Dnmt1 [black triangles; 0.125-2 µg plasmid DNA per transfection]. xSp1 activation of the reporter alone was assigned 100%. Data were obtained from nine independent assays and normalised to TK-Renilla. (C) Left panel: both forms of human DNMT1 (hDNMT1WT and hDNMT1C1226Y) are expressed equally after transfection into 293T cells relative to PCNA and endogenous hDNMT1 (-plasmid). Right panel: N2A cells were transfected with T7xSp1 against low or high xDnmt1 levels (black bar), cell extracts were blotted with -T7. Tubulin was used as a loading control. (D) An xOct91 promoter (-111 to +343) reporter construct is repressed by co-transfection with 500 ng of xDnmt1, hDNMT1WT and hDNMT1C1226Y but not by the empty vector control. (E) Chromatin IP (ChIP) analysis shows recruitment of GFP-xDnmt1 to the non-methylated xOct25 and xCycD1 promoters, but not an -tubulin intron in A6 cells. Note the enrichment of GFP-xDnmt1 at both promoters using -GFP (lane 2) but not the control xDnmt1 lacking GFP (panel xDnmt1). Lane 1, input (1/20 used in IP); -ve, no antibody control. (F) Bar chart shows fold enrichment of GFP-xDnmt1 at the xOct25 (eightfold) and xCycD1 (4.5-fold) promoters compared with a non-tagged control and with Tubulin.

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