The regulation of Ten-eleven translocation proteins (methylcytosine modifiers) by methyl-CpG binding domain proteins (methylcytosine readers)

• Main Author: Zhang, Peng
• Format: Others
• Language: en
• Published: 2016
• Online Access: https://tuprints.ulb.tu-darmstadt.de/5696/7/Peng%20Zhang.pdf; Zhang, Peng <http://tuprints.ulb.tu-darmstadt.de/view/person/Zhang=3APeng=3A=3A.html> (2016): The regulation of Ten-eleven translocation proteins (methylcytosine modifiers) by methyl-CpG binding domain proteins (methylcytosine readers).Darmstadt, Technische Universität, [Ph.D. Thesis]

Cytosine modifications diversify the genome and allow cell differentiation by the action of cytosine modification readers and modifiers. The epigenetic information of 5-methylcytosine can be translated by cytosine modification readers, such as the methyl-CpG binding domain (MBD) proteins. The aberrant interactions of MBD proteins with 5-methylcytosine cause diseases like Rett syndrome and also decrease genome stability, thus, the levels of both MBD protein and its substrate 5mC must be precisely regulated. Although, 5mC can be modified by Ten-eleven translocation (Tet) protein to 5-hydroxymethylcytosine (5hmC), which affects the binding ability of MBD proteins to DNA, the interplay of MBD proteins, Tet1 proteins and their substrate is still unknown. Since post translational modifications of Mecp2, the founding member of MBD protein family have been described before, we initially focused on the effect of poly(ADP-ribosyl)ation of Mecp2 on chromatin structure and its DNA binding ability. We show that in mouse brain endogenous Mecp2 is poly(ADP-ribosyl)ated in vivo. Furthermore, we find that poly(ADP-ribosyl)ation of Mecp2 decreases its ability to cluster pericentric heterochromatin. Finally, we demonstrate that poly(ADP-ribosyl)ated Mecp2 decrease binding ability to heterochromatin DNA. To understand the regulation of Tet mediated 5mC oxidation, we focused on how Tet proteins convert 5mC to 5hmC. We developed and optimized methods to step by step detect processes involved in Tet oxidation, including Tet-DNA binding, 5mC flipping and 5mC oxidation. We show that the catalytic domain of Tet1 (Tet1CD) binds to DNA in a non-sequence specific manner. Furthermore, we were able to detect DNA base flipping induced by Tet1CD. Finally, our methods can be used to easily and sensitively detect Tet oxidation products. By using these methods, we next tested whether MBD proteins affect Tet mediated 5mC oxidation. We focused on the five best studied MBD proteins including Mbd1, Mbd2, Mbd3, Mbd4 and Mecp2. We show that Mbd1 enhances Tet1 mediated 5hmC formation by facilitating its localization to methylated DNA. Moreover, the CXXC3 domain of Mbd1 is necessary for this enhancement. Compared with Mbd1, we find that Mbd3 and Mbd4 do not affect Tet1 mediated 5mC oxidation. In contrast to Mbd1, we show that Mbd2 and Mecp2 as well as its subdomains MBD and IDTRD block Tet mediated 5hmC formation in a concentration dependent manner in vivo and in vitro. Moreover, binding of Mecp2 to DNA impairs the DNA binding ability of Tet1CD in vitro and thus, direct binding of Mecp2 to DNA is sufficient to effectively prevent Tet1 mediated 5mC oxidation. These results indicate that the binding ability of MBD proteins and Tet proteins to DNA is important for 5mC conservation and conversion, respectively. Finally, we focused on the biological consequences of MBD proteins and Tet proteins mediated 5mC conservation and conversion. In mouse cells, we find that the Tet oxidation product 5hmC is enriched in neurons of mouse model for Rett syndrome (Mecp2 knockout mice). Moreover, we find that Tet1 reactivates expression of major satellite repeats in the absence of Mecp2. In human cells, we show that Tet1 activates endogenous and ectopic long interspersed nuclear elements 1 expression and transposition and this activation can be repressed by Mbd2 and Mecp2 as well as its subdomains MBD and IDTRD. These results indicate that the fine balance between methylcytosine readers” and “erasers/writers” regulates transcriptional noise and genome stability.