Epigenetic modifications has been well recognized as the key regulators of gene expression and take part in a variety of biological processes such as carcinogenesis, embryonic development and cellular reprogramming. Recently, two progresses have been made in the field of epigenetic and gene regulation by the joint effort of HE Chuan’s lab from Chicago University and JIANG Hualiang’s lab from Shanghai institute of Materia Medica. Their research revealed regulation mechanism of two key players in epigenetic modification and gene regulation: thymine-DNA glycosylase (TDG), which takes part in the DNA demethylation process, and AgrA, a bacterial response regulator in oxidation sensing and quorum sensing.
Human Thymine DNA glycosylase (hTDG) could efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. In addition, TDG has been proven essential for transcriptional regulation and mouse embryonic development. However, how TDG specifically recognized the modified cytosine in C:G based pair in the context of vast number of normal C:G base pairs remained elusive. HE’s team has shown that TDG could bind to different substrate DNA with distinct affinities. Based on the crystal structure of TDG in complex with DNA containing 5caC obtained by HE’s team, Jiang’s team perform molecular dynamics simulations and binding free energy calculations to study the dynamic process of TDG binding different substrates. The computational results revealed that the electropositive part of TDG binding pocket could form strong polar interaction with the negatively charged moieties of modified cytosines such as 5caC and 5fC. Thus, via a well-organized carboxyl-binding pocket, TDG could specifically binds 5caC over other modified cytosines. The result has been published online on Nature Chemical Biology (2012, 328–330).
In another cooperative study focusing on the regulatory mechanism of bacterial transcriptional factor AgrA, the two research teams further unveil that the intramolecular disulfide switch possessed by the DNA-binding domain of AgrA plays critical roles in the quorum-sensing agr system. Biochemical and mass spectrometric analysis performed by the researchers in HE’s lab reveal that oxidation induces the intracellular disulfide bond formation between Cys-199 and Cys-228, thus leading to dissociation of AgrA from DNA. By employing a combined computational approach including molecular modeling and molecular dynamic (MD) simulations, researchers from Jiang’s team indicate that the disulfide bond formation generates a steric clash responsible for the abolished DNA binding of the oxidized AgrA. Moreover, the C228S mutant rather than C199S mutant could unfold the DNA binding domain of AgrA, implicating C199 might be indispensable for oxidation sensing while C228 is structurally important for the proper folding of AgrA, which coincides with related experimental observations. Together, the results show that oxidation sensing is a component of the quorum-sensing agr signaling system, which serves as an intrinsic checkpoint to ameliorate the oxidation burden caused by intense metabolic activity and potential host immune response. Findings from this study provide key insights into the transcriptional regulation in bacterial and may serve as a launching pad for the development of novel antibacterial drugs. The result has been published online on PNAS on May 8, 2012.
These studies were funded by NIH, the Stem Cell and Regenerative Medicine grant from the Chinese Academy of Sciences, the grant from NSFC, 973 and 863.
The binding mode between TDG and 5caC The disulfide switch in quorum-sensing agr system (Image by SIMM)
