RESUMEN
We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) â d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.
Asunto(s)
5-Metilcitosina/análogos & derivados , Metilación de ADN , ADN/química , ADN/metabolismo , Epigénesis Genética , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Sitios de Unión , Fenómenos Biofísicos , Biología Computacional , ADN/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
The glutenin fraction of wheat storage proteins consists of large polymers in which high- and low-molecular-weight subunits are connected by inter-chain disulfide bonds. We found that assembly of a low-molecular-weight glutenin subunit in the endoplasmic reticulum is a rapid process that leads to accumulation of various oligomeric forms, and that this assembly is sensitive to perturbation of the cellular redox environment. In endoplasmic reticulum-derived microsomes, low-molecular-weight glutenin subunits are subjected to hyper-polymerization, indicating that cytosolic factors play an important role in limiting polymer size. Addition of physiological concentrations of reduced glutathione is sufficient to maintain the original polymerization pattern of the glutenin subunits upon cytosol dilution. Furthermore, we show that a low-molecular-weight glutenin subunit can be glutathionylated in endoplasmic reticulum-derived microsomes, and that it can be directly reduced by glutathione in vitro. These results indicate that glutenin polymerization is sensitive to changes in the redox state of the cell, and suggest that the presence of a reducing cytosolic environment plays an important role in regulating disulfide bond formation in the endoplasmic reticulum of plant cells.
