Different inter-domain linker regions regulate the binding of UHRF1 and NP95 to histone H3
by Maria Tauber
Date of Examination:2016-06-17
Date of issue:2017-06-15
Advisor:Dr. Wolfgang Fischle
Referee:Dr. Wolfgang Fischle
Referee:Prof. Dr. Michael Kessel
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Abstract
English
Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1 in human, NP95 in mouse) is an important epigenetic regulator connected to apoptosis and tumor development. UHRF1/NP95 integrates different chromatin modifications and is essential for their maintenance throughout the cell cycle. UHRF1/NP95 comprises domains for interaction with histones of different modification states (TTD and PHD) and methylated DNA (SRA) coupled to a catalytically active E3 Ubiquitin ligase domain (RING). While multivalent binding of multidomain proteins to complex patterns of chromatin modifications is a hallmark of the histone code, the regulation of such factors has not been fully investigated. Emerging evidence indicates that the chromatin binding modules of UHRF1/NP95 do not act in isolation but in concert to orchestrate coordinated interaction with different histone and DNA modifications. Here, we dissected the molecular details of regulation of chromatin binding by UHRF1/NP95. Using intramolecular crosslinking analysis and NMR studies we show that flexible inter-domain linker regions establish contacts between the different chromatin-binding modules and globally direct the overall folding and spatial domain arrangement of the protein. These contacts are modulated by ligand binding (histones, DNA) as well as by the allosteric regulator phosphatidylinositol 5-phosphate (PI5P). Indeed, in vitro binding experiments imply that the unstructured linker regions of the protein can alter binding behavior of the chromatin interaction domains and therefore control specificity of UHRF1/NP95. Interestingly, we found that murine NP95 has two splice variants, which only differ in a nine amino acid insertion in one of the inter-domain linker regions. In agreement with our hypothesis, this insertion is sufficient to facilitate differential regulation of chromatin binding by the two isoforms. Overall, our results show that flexible inter-domain linkers are versatile tools to dynamically regulate the interplay of several domains. We therefore propose a general model of fine-tuned combinatorial readout of epigenetic modifications by certain multidomain proteins.
Keywords: UHRF1, histone H3 K9me3, DNA methylation, replication