Kombinatorisches Auslesen epigenetischer DNA- und Histonmethylierung durch ICBP90/UHRF1
Biochemie
Strukturbiologie
Zellbiologie
Zusammenfassung der Projektergebnisse
UHRF1/NP95 is a multidomain chromatin protein that is essential for DNA maintenance methylation. Its different domains have distinct histone and DNA modification binding properties, namely for H3K9me3, H3unmod and hemi-methylated DNA. How the different chromatin binding functions of UHRF1/NP95 are coordinated and regulated is unclear. In the first project phase we had shown that the phosphoinositide PI5P functions as an allosteric regulator of UHRF1/NP95 inducing H3K9me3 binding. In one part of the current project, we have now further refined the understanding of this interaction. We could show that UHRF1 interaction with phosphoinositides is not only highly specific for the phosphorylation status of the inositol head group but also highly selective for medium length, saturated acyl chains. PI5P establishes a conformational state of UHRF1 that enables the strongest interaction to any histone modification, H3K9me3 observed to date. Using biochemical, biophysical and structural approaches, we mapped the exact interaction interface on UHRF1 for PI5P thereby establishing a model of chromatin binding domain coordination mediated by bridged interdomain linker regions. Cellular studies imply a putative role for H3K9me3 binding of UHRF1-PI5P in DNA methylation. A mouse in vivo model of UHRF1/NP95 mutated in PI5P interaction shows pleotropic phenotypes in early development that indicate a possibly role for the UHRF1-PI5P-DNA methylation axis in imprinting. In the second part of the project, we analyzed two splicing variants of the mouse UHRF1 protein, NP95 that only differ in a short, 9 amino acids insertion in an interdomain linker region of the protein that connects the H3K9me3- and H3unmod-binding domains. We could show that this change affects the chromatin binding properties of the protein. Biophysical and structural analysis defined that the change in the linker region controls the access of the H3K9me-binding domain to its target as well as the interplay of this domain with the adjacent H3unmod-binding domain. Cellular studies implied that the different splicing variants of NP95 have distinct chromatin localization and putatively different functionality in maintaining DNA methylation in naïve and stem cells. Despite immense efforts, it has so far not been possible to deduce a three-dimensional structure model of UHRF1 or NP95. All crystallization trials of different proteins and with distinct ligands have failed. To overcome this problem, we have now established stable conditions for high resolution NMR, HDX-MS and SAXS measurements and we are in the process of obtaining structural models for UHRF1 in apo and PI5P bound state. Besides regulation by PI5P, our work pointed to alternative splicing as a mechanism regulating chromatin binding of UHRF1/NP95. We have made good progress in understanding the consequences of different splicing on the level of molecular regulation of the protein. More work is, however, required to obtain insights into the biology and function of the different splicing variants. Lastly, our findings point to a putative role of UHRF1-PI5P binding in DNA methylation and in particular in establishing or maintaining imprints of allele specific gene expression. Further studies are required to solidify this link and to explore its biological role. It still needs to be sorted out if PI5P is indeed a regulatory ligand of UHRF1 function or serves as a constitutive cofactor. To this end, we will need to refine existing experimental systems for PI5P perturbation and measurement. Completely new thinking and experimental concepts might be required to ultimately crack the nuclear PIP code.
Projektbezogene Publikationen (Auswahl)
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(2015) Conserved linker regions and their regulation determine multiple chromatin-binding modes of UHRF1. Nucleus 6:123-132
Tauber, M. and Fischle, W.
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(2015) The basal transcription complex component TAF3 transduces changes in nuclear phosphoinositides into transcriptional output. Mol. Cell 58:453-467
Stijf-Bultsma, Y., Sommer, L., Tauber, M., Baalbaki, M., Giardoglou, P., Jones, D.R., Gelato, K.A., van Pelt, J., Shah, Z., Rahnamoun, H., Toma, C., Anderson, K.E., Hawkins, P., Lauberth, S.M., Haramis, A.P., Hart, D., Fischle, W. and Divecha, N.
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(2016) Oxidative stress signaling to chromatin in health and disease. Epigenomics 8:843-862
Kreuz, S. and Fischle, W.
