Studies On Interaction Between DNMT3A And Symmetric Methylation Of Histone H4Arginine3(H4R3Me2s)

β-Thalassemia and Sickle cell diseases (SCD) are two of the most common categories of hematopoietic diseases, caused by mutations of amino acid sequences on β-globin, a component of human hemoglobin. These mutations result in the abnormal structure and subsequent impaired oxygen binding capacity of human hemoglobin, which exert serious threats to human health. Conventional treatments for anemia include blood transfusion, splenectomy, bone marrow transplantation etc., all of which are currently not satisfactory.Upregulating the expression of y-globin to replace mutated β-globin could greatly alleviate the symptoms of anemia, becoming a better choice for treatment. Therefore, understanding the molecular mechanism underlining human fetal (y) globin gene silencing is very important.Previously our group has demonstrated that on the β-gene locus, the protein arginine methyltransferase PRMT5symmetrically methylated histone H4arginine3(H4R3me2s). Then H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, methylating CpG sites at the y-gene promoter, resulting in gene silencing. DNMT3A, a member of DNMT3family, is responsible for de novo methylating DNA. It contains three domains:PWWP domain, ADD domain in N-terminal region, and C-terminal catalytic domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, mainly connects to chromatin. The ADD domain, which stands for ATRX-DNMT3-DNMT3L, contains a PHD motif. ADD domain is also named PHD domain in some papers. ADD domain mainly binds to modified hitones. C-terminal catalytic domain is the core part of DNMT3A, methylating CpG sites in DNA.Histone H4arginine3can be symmetrically or asymmetrically dimethylated by protein arginine methyltransferases PRMT5or PRMT1. Asymmetrically dimethylated histone H4arginine3, H4R3me2a, has a relationship with gene activation, invoved in adult β-globin gene activation, whereas symmetrically dimethylated histone H4arginine3, H4R3me2s, serves as a gene repressed marker, invoved in y-globin gene silencing.We have confirmed that the binding occurred between DNMT3A and H4R3me2s through the ADD domain of DNMT3A (ADD3A). The thesis is dedicated to study a detailed mechanism of this recognition. Based on computer simulation, we spotted five potentially important amino acid residues (Trp528, Asp529, Asp530, Asp531, Trp533) on ADD domain of DNMT3A in its recognition with H4R3me2s, and made site-specific mutations on these five amino acid residues. Then in vitro, we tested binding abilities between wild-type, mutated ADD domains and H4R3me2s. In vivo, we examined the expression of γ-globin, methylation levels on the promoter of γ-globin locus and interaction between DNMT3A and PRMT5in both wild-type and mutated DNMT3A stable overexpressed cells. We found that in vitro, a D529A mutation on the ADD domain of DNMT3A resulted in decreased binding to H4R3me2s. Accordingly, y-globin mRNA level was upregulated in the DNMT3A overexpressed cell line with this mutation, with a notably decreased DNA methylation level at the y-globin promoter, and lower interaction between DNMT3A and PRMT5. These results revealed that aspartic acid residue529in DNMT3A plays an important role in the recognition of H4R3me2s. In summary, on the one hand, our findings contributed to understanding the detailed role of DNMT3A on y-globin gene regulation mechanism; on the other hand, these findings will help study the crosstalk between DNA methylation and histone modification, and their down-stream modulations on genes.