The GATA-1 transcription factor is like the Tiger Woods of the erythroid cell lineage, topping the leaderboard and driving the expression of important developmental genes. Other molecular competitors, like the NuRD complex, are already on the course, but a new study shows how methylation of a single CpG is able to score a hole-in-one to prevent GATA-1 binding.
Researchers from the lab of Merlin Crossley (University of New South Wales, Sydney, Australia) found that the relationship between GATA-1 and DNA methylation is anything but par for the course. After using electrophoresis mobility shift assays to confirm that GATA-1 binds to CGATA, AGATA and TGATA motifs in vitro, the talented team found that binding is disrupted by hemi- or fully methylated CGATA probes.
After chipping through existing GATA-1 ChIP-seq datasets from murine erythroleukemia (MEL) and erythroblasts for peaks with the CGATA motif, the savvy scientists focused their short game on an enhancer/silencer in the second intron of the c-Kit gene.
Here’s what they found:
- The CGATA element is highly methylated in mouse haematopoietic stem cells, but lowly methylated in erythroid cells, which were separated by flow cytometry and measured with bisulfite pyrosequencing
- This corresponds to a known decrease in c-Kit expression as these cells differentiate
- GATA-1 binds to the second intron of c-Kit in in wildtype MEL cells, as confirmed by ChIP-qPCR
- CRISPR/Cas9 gene-editing of the CGATA motif into TTATA disrupts GATA-1 binding and increases c-Kit expression
- CRISPR/Cas9 deletion of Tet2 in a cell line with an inducible GATA-1 protein (G1E-ER4 cells) increases endogenous methylation of the CGATA motif and reduces binding of GATA-1
- It also decreases GATA-1 binding to a non-CGATA binding downstream, in intron 4, as measured by ChIP-qPCR
- Inducing GATA-1 in WT G1E-ER4 cells decreases c-Kit expression, but induction has a much weaker effect in Tet2-KO cells where the element is more highly methylated
- In CRISPR/Cas9 edited mice, whose CGATA element was converted to TGATA, there is an accumulation of megakaryocyte-erythroid progenitors and a decrease in c-Kit expression in these cells compared to wild-type
Overall, this article gives a much-needed mechanistic example of how the methylation of a single CpG can regulate gene expression. Senior author Merlin Crossley concludes with the outlook that “Understanding epigenetics helps us understand not only normal gene biology, but why gene therapy vectors are often turned off and how we might keep them on – and how we could turn others – like viral genes – off.”
Tee up to the original article in Nature Communications, May 2020.