Fei Wang

Pre-Doctoral Fellow

Research Summary 

DNA methylation is a key epigenetic regulator of embryogenesis and stem cell differentiation in mammals. Distinct genomic patterns of DNA methylation are important features of stem cell identity. TET(TET1/2/3) proteins have been recently identified as enzymes regulating active removal of the DNA methyl mark. In Drosophila, DNA methylation has been controversial and it is believed not to have a profound function in genome-wide regulation of gene expression. A recent whole genome sequencing study demonstrated low levels of DNA methylation in Drosophila embryos. It is independent of DNMT2, the only known candidate DNA methyltransferase in Drosophila, implying that a novel DNA methytransferase is yet to be discovered. Tet , demonstrated as demethylases in vertebrates, is an essential gene in Drosophila, and loss or depletion of Tet leads to lethality in late pupa stage. This observation argues that DNA methylation in Drosophila may have dynamic profiles and important functions in regulating gene expression and that 5hmc may play a role in epigenetic regulation in Drosophila. Investigating the binding pattern of TET protein will provide crucial insights in understanding the function of DNA methyaltion in Drosophila and may shed light on additional function of Tet in vertebrates. 

 

The Drosophila Tet gene encodes 6 alternative transcripts detected by deep sequencing. Four of them are the full length containing the CXXC domain (binding to DNA) and the catalytic domain. Two transcripts encode only the catalytic domain. In order to characterize the function of Tet in Drosophila, it is necessary to generate different alleles which are specific to the different transcripts. I have been using FRT-mediated site-specific recombination to make deletions which remove 1) the DNA binding domain of the long transcripts (TetDBD); 2) the short transcripts resulting in the mutant Tet2del , and 3) the 5’ end of all transcripts resulting in the mutant Tetnew. Among all three mutants, Tetnew displayed the most severe defect in pupal ovaries; oogenesis was arrested and the somatic cells overpopulated the germarium. This result suggested that Tet might also have an important function in ovary stem cells.

Position effect variegation (PEV) measures the chromatin state by eye color change of a w+ minigene inserted into heterochromatin. In a Tet /+ background, the expression of a w+ reporter inserted in the centromere or telomere of the 4th chromosome was significantly elevated compared to WT, suggesting that Tet has an effect on chromatin organization. But it is still unknown whether Tet functions through DNA hydroxymethylation or through other, additional functions, to control epigenetic changes. 

 

In order to investigate the binding pattern of TET, I constructed a transgenic line expressing a tagged Drosophila Tet CXXC domain. By expressing this transgene under the heatshock-GAL4 driver I found that transgenic flies died at pupal stage when heat shocked at first instar larval stage. This indicates a dominant-negative effect of CXXC domain overexpression. I also examined the binding pattern of CXXC-RFP-Myc on salivary gland chromosomes and co-stained with other chromatin marks which are reported to be co-localized with human TET1 like H3K4me, H3K27me, H3k27ac. The CXXC domain overlaps with these marks to different extents. I am now using this line to map DNA binding sites of Drosophila Tet, using a deep sequencing approach.