Supplementary Information for Structural and Mechanistic Insights into Cooperative Assembly of Dimeric Notch Transcription Complexes

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1 Supplementary Information for Structural and Mechanistic Insights into Cooperative Assembly of Dimeric Notch Transcription Complexes Kelly L. Arnett 1,2, Matthew Hass 3, Debbie G. McArthur 1,2, Ma. Xenia G. Ilagan 3, Jon C. Aster 2, Raphael Kopan 3, and Stephen C. Blacklow 1,2,4 1 Department of Cancer Biology, Dana Farber Cancer Institute, Boston MA Department of Pathology, Brigham and Women s Hospital, and Harvard Medical School, Boston MA Department of Developmental Biology, Washington University, St. Louis, MO, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston MA Correspondence should be addressed to Stephen Blacklow: sblacklow@partners.org

2 Supplementary Fig. 1. Structural comparison to ideal B-form DNA. (a, left) Superposition of the NTC dimeric structure on the primary CSL binding site of an ideal B-form model of HES1 DNA. Compared to ideal B-DNA, the HES1 DNA in this structure is under-twisted in the spacer and bent away from the dimer interface. (a, right) A 90 rotation of the dimeric NTC structure around the dimer interface axis. The NTC trimer at the secondary site, in the background, has a translucent surface. (b) Two copies of the NTC modeled onto ideal B-form DNA with two CSL bindings sites separated by 15 bp, 16 bp, or 17 bp, illustrating the degree of untwist required for binding to CSL sites with these spacings.

3 Supplementary Fig. 2. Responsiveness of different promoter elements to ligand induction of full-length Notch1 or the R1985A dimerization-deficient mutant. Induction of full-length Notch1 or R1985A by (a) CHO cells stably expressing DLL1 ligand and (b) immobilized DLL1-Fc. Luciferase reporter gene expression under control of the TP1, Hes1, Hes5, Hey2, and HeyL promoters are shown. Fold activation for each reporter was calculated by normalizing to the cells transfected with CS2+ vector and treated with the ligand control (i.e., immobilized IgG or CHO cell coculture). Error bars represent standard deviations.

4 Supplementary Fig. 3. Cooperative dimerization of different NTCs on SPS DNA. (a) Mouse Hes5 E-site oligonucleotide duplexes used for Electrophoretic Mobility Shift Assay. (b) EMSAs performed on Hes5 SPS element using wild-type Notch1 RAMANK or mutant forms: R1985A, K1946E, E1950K, or K1946E E1950K. A low (50 nm) and a high concentration (200 nm) of CSL were incubated with labeled DNA, with an excess of wild-type or mutant RAMANK in the presence (top gel) or absence (bottom gel) of an excess of MAML. In the absence of MAML, wild-type and mutants forms of Notch all supershift CSL DNA complexes on a single site. In the presence of MAML, wild-type and the K1946E E1950K double mutant supershift to a higher order dimeric complex more effectively than the single mutants, R1985A, K1956E or E1950K.

5 Methods for Supplementary Fig. 2 DNA constructs The pcs2+n1fl R1985A vector was generated by subcloning from the N1 E R1985A plasmid into a pcs2+n1fl wt construct using SgrA1 and BglII sites. The TP1-Luc (pga981-6) reporter was a gift from Dr. Tasuku Honjo 1. To generate the pcdna4-delta1-igg expression construct, we replaced the B7 coding region within the B7-IgG vector (a gift from Dr. Kenneth Murphy; 2 ) with that of the mouse Delta1 extracellular domain. The resulting construct encodes aa1-535 of mouse Delta1 fused to the CH2-CH3 domain of mouse IgG1 and a Myc-His epitope tag. A corresponding control vector pcdna4-igg was also prepared by replacing B7h with the signal sequence of mdelta1 (aa1-25). Cell lines and culture conditions The CHO-GFP and CHO-DLL1-IRES-GFP stable lines were maintained in 10% FBS/IMDM (see 3 for details on their generation). 3T3 cells were maintained in 10% BCS/DMEM while 293T cells were maintained in 10%FBS/DMEM. Delta1-IgG production and immobilization To obtain conditioned media containing IgG or Delta1-IgG, 293T cells were seeded in P100 dishes, and transfected the next day with 10µg pcdna4-ssigg or pcdna4-delta1-igg expression plasmid using Fugene6 (Roche). After 24 hrs, the cells were fed with fresh 10%FBS/DMEM and incubated for another 48 hrs. Conditioned media were collected, filter-sterilized and stored at 4 C. Expression and secretion of the IgG and Delta1-IgG fusion proteins were confirmed by SDS-PAGE/Western analyses. To immobilize Delta1-IgG, affinity-purified anti-mouse IgG antibodies, Fc fragment specific (Jackson Immunoresearch; 10µg/ml in PBS) were initially adsorbed onto culture plates for 1 hr at 4 C. The plates were then washed once with PBS and incubated with conditioned media (diluted 1:4 with fresh medium) for 1 hr at 4 C. Prior to cell seeding, the conditioned medium was removed and the plates were washed once with PBS and equilibrated to RT.

6 Ligand-dependent Transcriptional Reporter Assays 3T3 cells were seeded at a density of cells/well in 24-well plates with and without immobilized ligand. The next day, the cells were transfected using Calcium phosphate with 200ng reporter plasmid (TP1-, HES1-, Hes5-, Hey2- or HEYL-Luc), 100ng CS2+ or CS2+N1FLwt or CS2+N1FL R1985A expression plasmid and 10 ng CS2+cytoβgal, to have 310 ng total DNA/well. After 18hr, transfected cells were fed with fresh medium. For the co-culture assays, CHO and CHO-DLL1 cells were trypsinized and resuspended in fresh medium and added (1.5x10 5 /ml) to the transfected 3T3 cells. After another 24 hrs, cells were lysed and analyzed for Luciferase and β-galactosidase activities as described 4. β- galactosidase was used to correct for differences in transfection efficiency. Fold activation for each reporter was calculated by normalizing to the cells transfected with CS2+ vector and treated with the ligand control (i.e., immobilized IgG or CHO cell coculture). 1. Minoguchi, S. et al. RBP-L, a transcription factor related to RBP-Jkappa. Mol Cell Biol 17, (1997). 2. Watanabe, N. et al. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1. Nat Immunol 4, (2003). 3. Ong, C.T., Sedy, J.R., Murphy, K.M. & Kopan, R. Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition. PLoS One 3, e2823 (2008). 4. Saxena, M.T., Schroeter, E.H., Mumm, J.S. & Kopan, R. Murine notch homologs (N1-4) undergo presenilin-dependent proteolysis. J Biol Chem 276, (2001).