Stancheva I and Meehan RR (2000), Transient depletion of xDnmt1 leads to prematur...

XB-ART-11499

Genes Dev 2000 Feb 01;143:313-27.

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Transient depletion of xDnmt1 leads to premature gene activation in Xenopus embryos.

Stancheva I , Meehan RR .

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In Xenopus laevis zygotic transcription begins at the midblastula transition (MBT). Prior to this the genome is organized into chromatin that facilitates rapid cycles of DNA replication but not transcription. Here we demonstrate that DNA methylation contributes to the overall transcriptional silencing before MBT. Transient depletion of the maternal DNA methyltransferase (xDnmt1) by anti sense RNA during cleavage stages is associated with a decrease in the genomic 5-methyl-cytosine content and leads to the activation of zygotic transcription approximately two cell cycles earlier than normal. Hypomethylation allows the early expression of mesodermal marker genes such as Xbra, Cerberus, and Otx2, which are subsequently down-regulated during gastrulation of the xDnmt1-depleted embryos. The temporal switch in gene expression may account for the appearance of body plan defects that we observe. Loss of xDnmt1 can be rescued by the coinjection of mouse or human Dnmt1 protein. These results demonstrate that DNA methylation has a role in the regulation of immediately early genes in Xenopus at MBT.

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Species referenced: Xenopus laevis
Genes referenced: cer1 dnmt1 gal.2 hdac3 hoxb3 hoxb9 otx2 pcna tbx2 tbxt
???displayArticle.antibodies??? Dnmt1 Ab2 Methyl Cytosine Ab2

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	Expression of xDnmt1 during Xenopusdevelopment. (A) The functional domains of the xDnmt1 protein are shown as shaded and solid boxes. (NLS) Nuclear localization signal; (RF) region involved in targeting of the enzyme to replication foci; (Zn) cystine-rich Zn/DNA-binding motif; (PBHD) polybromo-1 protein homologous domain; I are the conserved motifs of the methyltransferase catalytic domain. The 1.4-kb partial somaticxDnmt1 cDNA (st. 202) is 98% homologous to the oocytexDnmt1 catalytic domain (red). The blue bar indicates the PCR product used to detect xDnmt1 depletion in Figure 3A. (B) Northern blot analysis of xDnmt1 transcripts during embryo development. Ornithine decarboxylase (ODC) is an ubiquitously expressed gene. Stages of development are indicated above the xDnmt1 blot. (C,D) Whole-mount in situ hybridization localizes xDnmt1 transcripts (pink) to the animal pole of albino Xenopus egg (C) and animal pole blastomeres of 64-cell blastula (D). (An) Animal pole; (Veg) vegetal pole. (E) xDnmt1 is not detected in the vegetal hemisphere of 64-cell blastula. (F) After MBT the somatic form of xDnmt1 appears in the deep cells of the dorsal (DM) and ventral (VM) mesoderm of stage 11 gastrula (sagittal section). (G) During neurulation at stage 15 the staining forxDnmt1 transcripts is localized in the eye (e) and brain regions (br) of the prospective head neuroectoderm (pne) and along the edges of the open neural fold (nf). (H,I) A similar pattern is maintained during tailbud stages 20 (F) and 23 (G). (fb) Forebrain; (mb) midbrain; (hb) hindbrain. (J) At stage 35 (tadpole) the methyltransferase probe stains eyes, brain regions, branchial arches (ba), posterior notochord (nch), and the motor neurons (mn). (K) xDnmt1 protein (xDnmt1p) was immunoprecipitated from extracts derived from animal or vegetal halves of 64-cell blastulae and detected by Western blot analysis. Both extracts contain equal amounts of PCNA (bottom).
	Figure 2. Phenotypes of sense and antisense xDnmt1RNA-injected embryos. (A) Table showing the phenotypes of stage 35 embryos after injection of synthetic xDnmt1 RNA. (B) Four control embryos (stage 12.53) undergoing normal gastrulation and almost closing the blastopore (indicated by arrow). (C) The injection of 520 pg of antisense xDnmt1 RNA into the animal pole at two-cell stage slows gastrulation and disturbs the convergent extension movements of ectomesoderm. Shown are four representative gastrulae at the equivalent of stage 12.53. Note the extended appearance of the embryos and the large blastopore (indicated by arrow). (D) Uninjected control embryos at stage 35. (E) Injection of 520 pg of sense xDnmt1 RNA into two- and four-cell blastulae or 400 pg of antisense RNA into the vegetal pole does not lead to any visible developmental abnormalities. (F) The injection of a low dose antisense RNA (400 pg) into the animal pole of two- and four- cell blastulae causes the embryos to develop microcephally as shown at the equivalent of stage 35. (G) The injection of a high concentration (52000 pg) of the antisense RNA leads to considerable shortening of the dorsal axis in addition to microcephally.
	Figure 3. Microinjection of antisense xDnmt1 RNA transiently depletes the maternal xDnmt1 transcripts and protein during cleavage stages. (A) RT-PCR analysis ofxDnmt1 in RNA samples from wild-type blastulae (WT st.5) and antisense RNA-injected blastulae (AS st.5), wild-type (WT st.11) gastrulae, and antisense-injected (AS st. 11) gastrulae. The location of RT-PCR product with respect to xDnmt1 sequences and the antisense RNA is shown in Fig. 1A. Histone H1 (Hist H1) transcripts are present in equal amount in all RNA samples. (B) Protein extracts derived from wild-type (WT) and antisense RNA-injected (AS) embryos as in A were analyzed for the presense ofxDnmt1 by a carboxy-terminal polyclonal antibody. PCNA immunodetection is a loading control. (C) In situ hybridization with a 5â€² xDnmt1 probe confirms that thexDnmt1 transcripts (purple) are missing from the antisense-injected stage 6 blastula (AS) in comparison to a control embryo (WT) at the equivalent stage (viewed from the animal pole). (D) Sagittal section after in situ staining (magenta) with 5â€² xDnmt1 probe of stage 10 embryos shows that the zygoticxDnmt1 transcript is overproduced during gastrulation in the maternal xDnmt1-depleted embryo (AS) in the same region as in the control gastrula (WT). Dorsal is to the right; (bp) blastopores.
	Figure 4. Cross-species Dnmt1 proteins can substitute for the function of maternal xDnmt1 depleted by antisense RNA injection. (A) Microcephalic and axis-truncated phenotype of xDnmt1-depleted embryos can be rescued in a dose-dependent manner by coinjection of recombinant human (hDnmt1) or mouse (mDnmt1) Dnmt1 proteins with 520 pg of antisense xDnmt1 RNA. The percentage of resulting abnormal embryos scored at stage 35 was plotted for the experiments indicated underneath the bars. (n) Number of injected two-cell blastulae. Yellow indicates injection of antisense only; blue indicates injection of antisense plus recombinant hDnmt1p protein; light blue indicates injection of antisense plus recombinant mDnmt1p protein. (B) Both proteins (4 pg) cannot rescue the microcephalic phenotype. (C) Coinjection with 12 pg of human or mouse Dnmt1protein in the majority of cases can restore the normal appearance. (D) The rest of the embryos have normal or even enlarged heads, but still a very short axis. (E) hDnmt1 protein (40 pg) coinjected with the antisense RNA results in no-axis phenotype similar to that caused by injection of 12 pg of hDnmt1 protein alone into the animal pole at two-cell stage. (F) Vegetal pole cells are sensitive to the same amount (12 pg) of hDnmt1 protein injected alone. Vegetal injection into two-cell blastulae leads to multiple defects as shown at stage 35.
	Figure 5. Changes in the levels of 5-methylcytosine (m5C) during development of wild-type andxDnmt1-depleted embryos. (A) m5C was detected in the DNA of wild-type (WT) and xDnmt1-depleted (AS) embryos at the indicated stages (from egg to stage 35 tadpole). The antibody does not recognize the nonmethylated S. cerevisaeDNA (Y DNA). Note that there are higher levels (approximately three fold) of m5C in the DNA that derives from the animal pole blastomeres (An) than in the DNA from the vegetal cells (Veg). (X.bl)Xenopus blood and (X. sp) Xenopus sperm DNA samples. (B) The blot shown in A was stripped from the antibody and rehybridized with a mixture of Xenopus andS. cerevisae DNA to illustrate the equal DNA loading. (C) The relative amount of m5C in DNA of normal (WT) and xDnmt1-depleted (AS) staged Xenopus embryos as detected by the antibody in A in approximate units value. The wild-type oocyte DNA sample was taken as a standard (100%).
	Figure 6. xDnmt1-depleted embryos initiate zygotic transcription before MBT. (A) The incorporation of microinjected (50 nCi) [Î±-35S]UTP was used to detect the activation of gene expression in wild-type (WT) and sensexDnmt1 RNA (S) injected embryos. Wild-type blastulae were grown also in the presense of Î±-amanitin for the indicated amounts. (B) Embryos injected with antisense RNA and 50 nCi [Î±-35S]UTP (AS) at two-cell stage were cultured in parallel to the wild-type siblings and collected at the same time points as in A indicated on the graph. (C) RNA derived from wild-type (wt) and antisense-injected (as) embryos at stage 7 was assayed by RT-PCR for the transcripts of the mesodermal marker genes (Cerb, Xbra, Otx2), chromatin-related proteins (RPD3, HP-1Î± and Î³) and ubiquitously expressed EF1Î± (D) RT-PCR analysis of normal (wt) and xDnmt1-depleted (as) embryos at stage 11.5. Cerberus, Xbra, and Otx2 are down-regulated in the xDnmt1-depleted gastrulae. RPD3and HP1Î± are present at higher than normal levels. (E) RT-PCR analysis of normal (wt) andxDnmt1-depleted (as) embryos for the expression of the homeobox containing genes HoxB3 and HoxB9, neural Î²-tublin (NÎ²tub), muscle-specific actin(Mact), and histone H4 (Hist H4). (F) Antisense gastrulae rescued with 12 pg of mouse Dnmt1 protein (asâ€‰+â€‰hDnmt1) contains similar to wild-type (wt) levels ofCerberus, Xbra, xDnmt1, RPD3, and histone H4 transcripts. The expression of Otx2 always remained lower in the WT embryos. In C andF, âˆ’RT is a control PCR reactions of the antisense embryo RNA sample without reverse transcription.
	Figure 7. Localization of mesodermal markers in xDnmt1-depleted embryos before and after MBT. (A) Cerberus transcripts were detected by in situ hybridization in xDnmt1-depleted albino blastulae at stage 6.5. The albino embryos were injected symmetrically withxDnmt1 antisense RNA (as shown on the drawing).Cerberus transcripts localize predominantly to the most anterior animal blastomeres (purple staining). Animal view of the embryo. (B) Xbra transcripts (purple) in the antisense blastula of the same stage (6.5) appear around the midline zone (dorsal and ventral) and never overlap with the pattern ofCerberus expression. Animal pole view of the blastula. (C) Neither of the two transcripts are aberrantly expressed in the vegetal pole blastomeres of the antisense injected embryos (hybridization with Xbra is shown). (D) Wild-type blastula at stage 6.5 do not express Cerebrus or Xbra(control hybridization with Cerberus). (E) Ectopic injection of antisense RNA and Î²-gal sense RNA into a single dorsal animal blastomere (D1) of eight-cell blastulae (as indicated on the drawing) depletes xDnmt1 transcripts in a portion of animal pole cells. Only the staining for xDnmt1 is shown (red). (F) Few cells of the dorsal midline zone express ectopically high levels of Xbra (purple). (G) The ectopicXbra expression (dark blue) colocalizes with the staining for Î²-gal (blue) on the injected site of a stage 6.5 blastula. Note that Cerberus and Xbra expression localize to different populations of cells. The staining forxDnmt1 is in red. (H) Uninjected embryo at stage 6.5 does not express Xbra or Cerberus. (I) A few of the most anterior animal cells that coincide with the site ofxDnmt1 depletion express high levels of Cerebrus(purple) in a stage 6.5 blastula. (J) The staining forCerebrus transcripts colocalizes with that of the Î²-gal tracer. (K) The control embryo at the same stage (6.5) injected with Î²-gal RNA only do not show any staining forCerberus or Xbra (control hybridization with Cerebrus probe). (L) In xDnmt1-depleted embryo (AS) at stage 12 the staining for Cerberus transcripts is reduced compared to a wild-type (WT) gastrula (Cerebrus in dark brown to black). The remaining transcripts still localize to the most anterior regions of dorsal mesoderm. (M) Xbra (dark brown to black staining) is dramatically reduced in xDnmt1-depleted gastrula at stage 12.5 (AS) as compared with the wild-type (WT) embryo.
	Figure 8. Sequences of Xbra promoter are hypomethylated earlier in xDnmt1-depleted embryos. (A) Schematic presentation of 1.6-kb region that covers the promoter and the first exon of Xbra gene. The CpG pairs in the sequence are shown by vertical lines. Underneath the CpG plot are indicated HpaII and HhaI sites. The position of transcriptional initiation is marked by an arrow. Sequences that were analyzed by methylation-sensitive PCR during development are shown on the expanded region below. A 470-bp PCR product covers the sequences between âˆ’267 and +203 where there are two HhaI (Hh) sites and one HpaII (H) site. The 470-bp band is produced only if the methylation-sensitive enzymes HpaII and HhaI do not cleave in these sites. When the restriction sites are cleaved it results in the appearance of 249-bp PCR product and is indicative of hypomethylation (see Materials and Methods). Digestion withMspI (which is not methylation sensitive) gives rise to the shorter product only. (B) To create a quantitative standard curve, a plasmid containing the Xbra promoter and part of the first Xbra exon (Latinkic et al. 1997) was methylated in vitro by SssI methylase and mixed with nonmethylated Xbraplasmid in the combinations shown. The mixtures were digested withHpaII and HhaI and used for PCR amplification resulting in fragments of 470 and 249, respectively. The percentage of methylation is indicated on top of each lane. M is a 100-bp DNA marker. (C) Standard curve generated from plotting the ratio of 470/249 fragments (X axis) against the percentage of DNA methylation (Y axis). (D) Genomic DNA samples from staged embryos indicated by numbers from 40 (stage) were digested withHhaI and HpaII and analyzed by methylation-sensitive PCR. (E) Genomic DNA samples of the xDnmt1-depleted embryos at equivalent stages to the wild type in B, indicated by numbers from 4 to 35, were digested with HhaI and HpaII, and subjected to PCR analysis as in B. (F) The same of set of DNA samples as in D was digested with MspI and analyzed as above. (G) Graphical plot of methylation changes in Xbra promoter during development of the normal (WT) and xDnmt1-depleted (AS) embryos. The ratio of 470-bp to 249-bp PCR products for each sample was quantitatively estimated and plotted against a standard curve to give the amount of methylated Xbra promoter as a % of the totalXbra promoter sequences present in the PCR reaction of genomic DNA at the indicated stages.
	dnmt1 (DNA (cytosine-5-)-methyltransferase 1 ) gene expression in Xenopus laevis unfertilized egg as assayed by in situ hybridization. Equatorial view, animal up.
	dnmt1 (DNA (cytosine-5-)-methyltransferase 1 ) gene expression in Xenopus laevis embryo as assayed by in situ hybridization, NF stage 35. Lateral view: Dorsal up, anterior left

References [+] :

Almouzni, Constraints on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis. 1995, Pubmed, Xenbase