A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes

Published online 23 February 2016 Nucleic Acids Research, 2016, Vol. 44, No. 10 4703–4720 doi: 10.1093/nar/gkw105 A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes Franz Oswald1,*,† , Patrick Rodriguez2,† , Benedetto Daniele Giaimo3,4,† , Zeus A. Antonello5 , Laura Mira5 , Gerhard Mittler6 , Verena N. Thiel1 , Kelly J. Collins7 , Nassif Tabaja7 , Wiebke Cizelsky8,9 , Melanie Rothe8,9 , Susanne J. Kühl8 , Michael Kühl8 , Francesca Ferrante3 , Kerstin Hein3 , Rhett A. Kovall7 , Maria Dominguez5 and Tilman Borggrefe3,* 1 Received October 23, 2015; Revised January 22, 2016; Accepted February 11, 2016 ABSTRACT The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2␤. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo. Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier. INTRODUCTION A common feature of major cell-cell signaling pathways, such as Wnt, Hedgehog and Notch, is a shift from repression to activation of target gene expression that is mediated by the same transcription factor. Gene repression and activation are normally accompanied by specific chromatin modifications that involve dynamic changes in histone tail modifications, including acetylation and methylation (1–3). However, the interplay between chromatin modifiers and signal-dependent transcription factors is poorly understood. Notch signaling is one of a few signaling pathways that is used iteratively during development and adult tissue homeostasis (4–8). Notch-mediated transcriptional re- * To whom correspondence should be addressed. Tel: +49 641 9947400; Fax: +49 641 9947409; Email: Correspondence may also be addressed to Franz Oswald. Tel: +49 731 50044544; Fax: +49 731 50044542; Email: † These authors contributed equally to the paper as first authors.  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact University Medical Center Ulm, Center for Internal Medicine, Department of Internal Medicine I, Albert-Einstein-Allee 23, 89081 Ulm, Germany, 2 Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland, 3 Institute of Biochemistry, University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany, 4 Spemann Graduate School of Biology and Medicine (SGBM), Faculty of Biology, Albert Ludwigs University Freiburg, Germany, 5 Instituto de Neurociencias, Consejo Superior de Investigaciones Cientificas-Universidad Miguel Hernández, Campus de Sant Joan, Alicante, Spain, 6 Max-Planck-Institute of Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany, 7 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267, USA, 8 Institute for Biochemistry and Molecular Biology, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany and 9 International Graduate School in Molecular Medicine Ulm (IGradU), Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany 4704 Nucleic Acids Research, 2016, Vol. 44, No. 10 tone acetylation sites (27). Notably, H3K4 trimethylation is dynamically controlled at Notch target genes by histone demethylases LSD1 (28) and KDM5A/RBP2/JARID1A (29) and a hitherto unknown methyltransferase. The H3K4 methylation mark is written by enzymes of the KMT2 (lysine methyltransferase 2) family, which are parts of different large multisubunit complexes (30). All of them share the core subunits WDR5, RbBP5 and Ash2l but whereas Menin1 is a specific subunit of the KMT2A/B complexes, UTX, PA1, PTIP and NcoA6 are specific subunits of the KMT2C/D complexes (31–33). Here, we show that the RBP-J associated cofactor SHARP interacts with either KMT2D coactivator or NCoR corepressor complex regulating the chromatin environment at Notch target genes. KMT2D and NCoR competitively bind to the same region of the cofactor SHARP, its SPOC domain. NCoR/KMT2D balance depends on the phosphorylation status of two conserved serine residues at the C-terminus of NCoR. Our data suggest that the conversion of RBP-J from a repressor to an activator is not a simple single-step process, as previously thought, but involves a chromatin intermediate directed by cofactor SHARP. The novel dual functionality of the RBP-J/SHARP complex may facilitate a robust, yet flexible, Notch-dependent transcriptional response. MATERIALS AND METHODS Cell culture and preparation of cell extracts Murine pre-T lymphoma cell line (Beko) and murine hybridoma mature T-cell line (MT) were grown in Iscove’s Modified Dulbecco Medium (Gibco) supplemented with 2% FCS, 0.3 mg/l peptone, 5 mg/l insulin, nonessential aminoacids and penicillin/streptomycin. Cells were grown at 37◦ C with 5% CO2 . pre-T cells were treated with 10 ␮g/ml DAPT/GSI (Alexis ALX-270–416-M025) or DMSO as control. Mature T-cells were treated with 25 ␮M TBB (4,5,6,7-tetrabromobenzotriazole, Sigma-Aldrich T0826), 25 ␮M TBCA [(E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid, Millipore 218710] or DMSO as control. Cell lines HEK293 (ATCC CRL 1573), 293 T and HeLa (ATCC CCL 2) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin. For western blotting, EMSA and immunoprecipitation experiments whole-cell lysates were prepared as previously described (20). Protein concentrations were determined using the Bradford assay method (BioRad). Streptavidin-immunoprecipitation of bio-SPOC domain of SHARP The SPOC-domain of SHARP containing an N-terminal biotinylation tag (34) (...truncated)