Mechanistic and functional insights into the recognition and regulation of DNA modifications by UHRF1, DNMT1 and TET proteins

The regulatory epigenome is essential in the development of organisms as it greatly contributes to the establishment and maintenance of cellular identity. Different layers of epigenetic control, for instance the chemical modification of histones and DNA, are closely interconnected and determine the accessibility of chromatin and how genetic information is utilized in different cell types. These layers stably protect genome integrity on the one hand and enable a certain degree of phenotypic plasticity on the other as they dynamically respond to external stimuli and environmental changes. This thesis aimed to further examine how DNA methylation patterns are regulated within the epigenetic landscape and to dissect the precise function of proteins directly involved in controlling DNA methylation levels, especially UHRF1, DNMT1 and TET proteins. In contrast to other epigenetic marks, the inheritance of DNA methylation patterns is well-studied and relies mainly on the activity of the maintenance methyltransferase DNMT1 and its co-factor UHRF1. Within this thesis, a systematic in vitro analysis of the binding properties of UHRF1 towards different DNA modifications is described, revealing that UHRF1 exhibits a preference for carboxylated cytosine (caC) besides hemi-mC. This is based on specific binding modes and the highly flexible NKR finger region of UHRF1 as investigated in complementary MD simulations. Furthermore, UHRF1 is shown to generate a second recruitment signal for DNMT1, namely ubiquitylated PAF15 (PAF15ub2), which is similarly bound by DNMT1 as H3K9Ub2. Whereas maintenance methylation through DNMT1 in early S-phase is demonstrated to mainly dependent on PAF15Ub2, H3Ub2 is important for the methylation of late-replicating chromatin. Additionally, the investigation of naïve pluripotent mESCs uncovered that the hypomethylated genome, characteristic for these cells, is largely promoted by the inhibition of the maintenance methylation machinery through DPPA3-mediated abrogation of UHRF1 binding to chromatin. It is further described that the expression of DPPA3 is directly regulated by TET1 and TET2, two α-ketoglutarate-dependent dioxygenases, which actively remove methylation marks, and that this DPPA3-mediated passive demethylation represents an evolutionary new concept of boreoeutherian mammals. Another section of this thesis addresses the metabolic regulation of TET proteins in mESCs and demonstrates that α-ketoglutarate constitutes a rate-limiting factor for the activity of these enzymes with consequences on pluripotency. Moreover, the inhibitory effect of 2-HG, an oncometabolite produced by mutant IDH enzymes, is also examined in mESCs, offering the possibility to precisely study the basis of epigenetic alterations observed in tumors harboring IDH mutations. Lastly, this thesis includes the examination of cross-regulating functions of TET1 and mC-binders, in particular MeCP2 and MBD2. As evident in vitro and in vivo, mC-binding proteins restrict the access of TET1 to DNA thereby protecting methylated cytosines from TET1-mediated oxidation. This in turn is discussed to be a critical mechanism lacking in patients with Rett syndrome, a neurological disorder caused by MeCP2 mutations. In conclusion, this work provides mechanistic and functional insights into the role of UHRF1, DNMT1 and TET enzymes in recognizing and regulating DNA modifications and highlights new aspects of these factors during mammalian development and disease