Abstract
Alterations in DNA methylation occur during development, but the mechanisms by which they influence gene expression remain uncertain. There are few examples where modification of a single CpG dinucleotide directly affects transcription factor binding and regulation of a target gene in vivo. Here, we show that the erythroid transcription factor GATA-1 — that typically binds T/AGATA sites — can also recognise CGATA elements, but only if the CpG dinucleotide is unmethylated. We focus on a single CGATA site in the c-Kit gene which progressively becomes unmethylated during haematopoiesis. We observe that methylation attenuates GATA-1 binding and gene regulation in cell lines. In mice, converting the CGATA element to a TGATA site that cannot be methylated leads to accumulation of megakaryocyte-erythroid progenitors. Thus, the CpG dinucleotide is essential for normal erythropoiesis and this study illustrates how a single methylated CpG can directly affect transcription factor binding and cellular regulation.
Highlights
Alterations in DNA methylation occur during development, but the mechanisms by which they influence gene expression remain uncertain
We extend these observations by showing that a single point mutation that converts the CGATA element to a TGATA site in a regulatory region of c-Kit—which can still be bound by GATA-1 but that is no longer sensitive to methylation—interferes with normal haematopoiesis in mice
Using an electrophoretic mobility shift assay (EMSA) we confirmed that COS cell overexpressed and murine erythroleukemia (MEL) cell endogenous GATA-1 is able to bind to CGATA, AGATA and TGATA, but less well to GGATA motifs, as previously reported[20] (Fig. 1b and Supplementary Fig. 1a)
Summary
Alterations in DNA methylation occur during development, but the mechanisms by which they influence gene expression remain uncertain. The CpG dinucleotide is essential for normal erythropoiesis and this study illustrates how a single methylated CpG can directly affect transcription factor binding and cellular regulation. We explored how DNA methylation at a single CpG dinucleotide could interfere with binding and regulation by GATA-110–13, a critical transcription factor that modulates the expression of most if not all erythroid-specific genes[14,15,16]. We show that methylation of a CGATA element reduces GATA-1 binding and gene regulation in cell lines We extend these observations by showing that a single point mutation that converts the CGATA element to a TGATA site in a regulatory region of c-Kit—which can still be bound by GATA-1 but that is no longer sensitive to methylation—interferes with normal haematopoiesis in mice
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