| As two important parts of epigenetics, histone modifications and DNA methylation play very important roles in the regulation of gene expression. They are involved in the binding of histone effector molecules, chromatin structure and embryonic development. As a genetic regulatory factor, UHRF1 can recognize both histone modification and DNA methylation. It has been shown to participate in a variety of biological processes, especially in the maintenance of DNA methylation. In the process of DNA replication, UHRF1 can recognize and bind to hemimethylated DNA, which further recruit DNA methyltransferase DNMT1 to catalyze methylation reactions on the newly synthesized DNA chain to maintain DNA methylation. Previous studies in our lab have demonstrated that the specific recognition of histone modifications by UHRF1 can affect its function on the maintenance of DNA methylation. But the recognition of histone modifications and DNA methylation by UHRF1 on nucleosome level is not clear. In this study, we investigate the interaction of UHRF1 with different kinds of nucleosome by in vitro reconstitution. First, we generate different point mutations in the TTD and PHD domain of UHRF1. Then we study the binding abilities of UHRF1 mutations with nucleosomes containing H3K9 methylation. The results show that both TTD and PHD domain are responsible for the specific binding of UHRF1 to histone H3K9 methylation. We also test the binding ability of UHRFl with hemimethylated DNA in different positions of the nucleosome. We generate nucleosomes without hemimethylated DNA and with hemimethylated DNA in five different positions. Then we study the binding abilities of UHRF1 with six different nucleosomes. Our results show that binding ability of UHRF1 is weaker when hemimethylated DNA position is closer to the internal core region of nucleosome. Our studies provide some in-depth information on the molecular mechanism of histone modification and DNA methylation by UHRF1 mediated. |
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