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. 2008 Aug 1;22(15):2075-84.
doi: 10.1101/gad.1658408.

Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b

Humaira Gowher  1 , Heidi Stuhlmann, Gary Felsenfeld
Affiliations

Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b

Humaira Gowher et al. Genes Dev. .

Abstract

The zinc finger protein vascular endothelial zinc finger 1 (Vezf1) has been implicated in the development of the blood vascular and lymphatic system in mice, and has been characterized as a transcriptional activator in some systems. The chicken homolog, BGP1, has binding sites in the beta-globin locus, including the upstream insulator element. We report that in a mouse embryonic stem cell line deletion of both copies of Vezf1 results in loss of DNA methylation at widespread sites in the genome, including Line1 elements and minor satellite repeats, some imprinted genes, and several CpG islands. Loss of methylation appears to arise from a substantial decrease in the abundance of the de novo DNA methyltransferase, Dnmt3b. These results suggest that naturally occurring mutations in Vezf1/BGP1 might have widespread effects on DNA methylation patterns and therefore on epigenetic regulation of gene expression.

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Figures

Figure 1.
Figure 1.
Loss of Vezf1 results in loss of DNA methylation. (A) Ethidium bromide stained 0.8% agarose gel loaded with genomic DNA from wild-type (W) and Vezf1−/− (V) ES cells. DNA was cleaved with HpaII (H), or MspI (M), or uncut (Schulz et al. 2002). (B) Methylation analysis by Southern blot; DNA was digested with HpaII and MspI and probed for different repetitive elements in the genome. MspI digestion was used as control to represent complete loss of DNA methylation. Line1 and Minor satellites show a distinct loss of methylation. A lesser loss of methylation is also seen at Sine B, telomeric, and major satellite repeats.
Figure 2.
Figure 2.
Methylation-dependent PCR analysis of sites at selected genes. Genomic DNA was cleaved with HpaII (H) and MspI (M). (A–C) Ethidium-bromide-stained 0.8% agarose gel loaded with PCR amplified cleaved genomic DNA from wild-type and Vezf1−/− ES cells. (A) Tissue-specific genes. (B) Imprinted genes. (C) Housekeeping genes. (D) Methylation analysis at H19 ICR and Igf2r DMR2 by bisulfite sequencing. The results show a significant loss of methylation at some sites (see the text).
Figure 3.
Figure 3.
(A) MeDIP/CpG island microarray analysis, comparing genomic DNA from wild-type and Vezf1−/− ES cells. Observed score (see the Materials and Methods) is plotted against expected score (based on randomized sample). The limiting parallel lines are chosen to give a false discovery rate of 29.7%. Analysis shows that almost all the CpGis with significant change (points lying outside the parallel lines) have lost DNA methylation (gray diamonds). Gray triangles represent gain of methylation. (B,C) RT–PCR analysis of genes selected from the microarray that showed significant loss of DNA methylation at CpGis were tested for change in expression. The asterisks indicate the genes that show increased expression in the Vezf1−/− cells.
Figure 4.
Figure 4.
Effect of Vezf1 deletion on expression levels of DNA MTases and of genes coding for other proteins known to modulate DNA methylation levels in the genome. (A,B) One-step RT–PCR was performed with total RNA from wild-type and Vezf1−/− cells to amplify DNA MTases, LSH, CGBP, Dnmt3 L, and β-actin as a control for the amount of RNA template used in the RT–PCR reaction.
Figure 5.
Figure 5.
Analysis of the splice variants of Dnmt3b; Dnmt3b transcript is decreased in Vezf1−/− cells. (A) Map of Dnmt3b gene showing three characterized splicing events. To analyze alternative splicing events, total RNA from wild-type and Vezf1−/− ES cells was reverse transcribed and amplified by PCR for 27 cycles. (B) Catalytic domain splicing. (C) 5′ end splicing. (D) Relative quantitative RT–PCR analysis for the measurement of DNA MTases using Taqman expression assay (Materials and Methods) The Ct values for all MTases are normalized to that of β-actin.
Figure 6.
Figure 6.
Transfection of Vezf1−/− ES cells with a Vezf1 expression construct restores DNA methylation. (A) Ethidium-bromide-stained gel of genomic DNA from Vezf1−/− ES cells transfected with a construct expressing Vezf1 and cleaved with HpaII (H) and MspI (M). Compared with untransfected Vezf1−/− cells, the transfected cells show less cleavage by HpaII, reflecting increased methylation of these sites. (B) DNA methylation analysis of repetitive elements in transfected cells: Southern blot of genomic DNA cleaved with HpaII and MspI and hybridized with probes against Line1 and minor satellite repeats. Both repeat elements show partial rescue of methylation in comparison with untransfected cells. (C) Relative quantitative RT–PCR analysis of Dnmt3b transcripts, normalized to β-actin as control using the TaqMan assay.
Figure 7.
Figure 7.
ChIP analysis of Vezf1/BGP1 binding and histone acetylation in vivo at sites in the Dnmt3b gene. (A) Map of the Dnmt3b gene with positions of the sites studied, showing oligo-G sequences that are potential Vezf1-binding sites. (B) ChIP of formaldehyde cross-linked chromatin from wild-type and Vezf1−/− ES cells with anti-Vezf1 antibody. DNA from IP fractions was used for quantitative PCR analysis at the Dnmt3b promoter and at the three oligo-G sequences. Fold enrichment over input is compared for wild-type and Vezf1−/− cells (see the Materials and Methods).
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