5 ZFHD sites in variable distances to transcription start site (Fig. S2) pmrlucf luciferase reniformis for Gal4 yeast pmrlucfpa 12Galn

Transcription

1 protein origin reference region (aa) construct comment Gal4 yeast -47 pmcgal4-47 pmcrluc-gal4-65 fusion of Rluc, Gal4(-47) and AD of artificial (0) pmczf s fused C-terminally to with 3x myc linker (EQKLISEEDLNG) or (GGGS) 7 pmcrluc-zf-65 fusion of Rluc, and AD of human FKBP2 human -07 pmceafk MinE (e) and acid (a); fused C-terminally with 3x myc linker (EQKLISEEDLNG) pmcafk; pmcafk2; pmcafkm; pmcafk6m for optimization of FK number (Fig. S3), pmcafkm corresponds to 3 FK domains FRAP human pmcfr-65 reporter origin reference region (aa) construct comment firefly Photinus pyralis pmlucf reporter plasmid with Fluc gene and minimal Fos promoter for Gal4 pmlucf [2-2]Gal4 2-2 dimeric Gal4 binding sites upstream of a minimal Fos promoter () for pmlucf [4-24]ZF 4-24 binding sites upstream of a minimal Fos promoter () pzfluc5m80; pzfluc5m200; pzfluc5m440; pzfluc5m800 5 sites in variable distances to transcription start site (Fig. S2) Renilla Renilla pmrlucf reniformis for Gal4 yeast pmrlucfpa 2Galn Gaussia Gaussia pmcglucs internal reference princeps origin reference region (aa) construct comment base artificial (3) pmczf-base pmczf-base[-6] pmczf-base[-7] pmczf-base[-9] pmczf-base[-0] MinD E. coli (22) full length pmcgal4-mind Lamin C human pmczf-lamin Lef human NM_ full length pmczf-lef Rb human NM_ full length pmczf-rb E-cadherin human NM_ pmczfg he-cadherin( c) Blimp human NM_ full length pmczf-blimp pmczf-base[-6]-blimp Pax5 human NM_ full length pmczf-pax5 pmczf-base[-6]-pax5 Gbx2 mouse NM_ full length pmczf-gbx2 pmczf-base[-6]-gbx2 Otx2 mouse NM_ full length pmczf-otx2 pmczf-base[-6]-otx2 En2 mouse NM_ full length pmczf-en2 pmczf-base[-6]-en2 Hes rat NM_ full length pmczf-hes pmczf-base[-6]-hes p53 human NM_ pmczf- p53(3-28) pmczf-base[-6]-p53(3-28) origin reference region (aa) construct comment acid artificial (3) pmcam; pmceafk pmcafk; pmc-acid-vp6 ß-catenin human NM_ pmc-βcat -VP6 pmc-β-cat -FK P peptide artificial (4) pmc-p-vp6; pmc P-FK Tle4 mouse NM_ full length pmceafk-tle4 Mdm2 human NM_ pmceafk-mdm2(25-3) MinE E. coli (22) -3 pmceafk Table S. Plasmids.

2 Rluc DBD 24x rel activity Gal transfected activator plasmid [ng] Figure S. Titration of binding domain and reporter construct optimisation. Cells were transfected with a reporter plasmid containing 24 binding sites of Gal4 (pmlucf 2 Galn, containing 2 dimeric Gal4 sites) or (pmlucf 24 ZF) together with varying amounts of corresponding activator plasmid (pmcrluc-gal4-65 or pmc Rluc-ZF-65). The y- axis shows relative activity as Fluc/Rluc relative to an empty reporter control without any binding site (pmlucf). Note that the values are corrected for expression level, increasing activity therefore represents non-linear synergistic increase. A combination of 24 binding sites and 2 to 20 ng expression construct (corresponding to the construct of the M2H) showed best results for these experiments. Similar results were obtained also for titration experiments of Gal4 and reporters for the M2H experiments. All data show the mean of at least 3 independent experiments containing 6 replicates each. Error bars indicate standard error.

3 rel activity [%] Rluc bp 220 bp transcription start site 460 bp 820 bp distance of binding sites from transcription start [bp] Figure S2. Effect of promoter size. Four different reporters containing 5 binding sites with a distance of bp (pzfluc5m80), 220 bp (pzfluc5m200), 460 bp (pzfluc5m440) and 820 bp (pzfluc5m800) between the transcription start site and the closest binding site were transfected into cells. To activate the reporter 0.2 ng activator plasmid containing binding domain fused to activation domain of and Rluc (pmcrluc-zf-65) for internal normalization were co-transfected. Dual was measured 24 hrs after transfection. Y-Axis shows relative expression in percent as Fluc/Rluc relative to the activator construct with the smallest distance between binding sites and transcription start site (pzfluc5m80). Values show the mean of 6 replicates. Error bars indicate standard deviation.

4 A FK FR FR FK RAP FR basal FK FK FR induced FK RAP FR FK RAP FR 24x 24x B C 0 base[-6] lamin 0 FR-65 FR-VP6 rel activity 0 rel activity x FK 2x FK 3x FK 6x FK acid base[-6] acid-fk lamin Figure S3. Optimisation of the AD. The influence of AD multimerization was studied by constructing proteins fused to different numbers of FK domains (, 2, 3 or 6). To test the optimal number of FK domains (B) cells were transfected with reporter (pmlucf 24ZF), construct containing base[-6] (dark grey bars) or lamin control (light grey bars) and construct containing acid fused to x, 2x, 3x or 6x FK domain and together with pmcfr-65. For comparison of different ADs (C) cells were transfected with reporter (pmlucf 24ZF), protein containing base[-6] (dark grey bars) or lamin control (light grey bars), and an acid construct (pmcafk) together with a FR construct either fused the AD of (pmcfr- 65) or VP6 (pmcfr-vp6). For internal normalization pmcglucs was co-transfected. Y- axis shows relative activity as Fluc/Gluc from rapalog treated relative to uninduced cells. All values show mean of at least 3 independent experiments with 6 replicates each. Error bars indicate standard error.

5 rel activity acid base VP6 24x full length [-6] [-7] [-9] [-0] ZF ZF-base lamin acid-vp6 K K W K Ac - AQ E E QA E E AQ E E QA E E AQ - NH2 L - - L L - - L L L L - - L L L L L L L L L Ac - AQ K K QA K K AQ K K QA K K AQ - NH2 K K W K acid base [-6] base [-7] base [-9] base [-0] base Figure S4. Analysis of artificial leucine zipper peptides as reference interactions. Truncated versions of the base peptide (3) were constructed deleting the last 6 (base [-6]), 7 (base [-7]), 9 (base [-9]) or 0 (base [-0]) amino acids resulting in the loss of to 3 central interactions of the leucine zipper structure. For analysis cells were transfected with reporter plasmid (pmlucf 24ZF), 20 ng of a plasmid expressing full length acid fused to the activation domain of VP6 (pmcamvp6) as protein and 20 ng of protein expressing full length base (pmczf-base) and base truncations (pmczf-base[-6], pmczf-base[-7], pmczf-base[-9], pmczf-base[-0]) fused to DBD, Lamin C functions as a negative control. 2 ng pmc GlucS was co-transfected as internal reference. Y-axis shows relative expression as Fluc/Gluc relative to negative control (ZF lamin). All values represent the mean of at least 3 independent experiments with 6 replicates each. Error bars indicate standard error.

6 rel activity 0 : Tle4 no MinE + MinE 0 0. En2 Gbx2 Pax5 Figure S5. Effect of MinE on interaction. To compare the effect of the N-terminal fusion of MinE to the protein, Tle4 interactions were analysed in the absence (no MinE, grey bars) or presence (+ MinE, orange bars, same data as shown in figure 3B, dual luc) of MinE fusions. For no-mine experiments cells were transfected with test reporter (pmlucf 24ZF) and constructs containing En2, Gbx2 or Pax5. The construct expresses Tle4 fused to 3 FK domains together with 20 ng pmcfr-65 and constitutive gaussia for normalization. Y-axis shows relative Fluc/Gluc (no MinE) or Fluc/Rluc (+ MinE) relative to lamin control. All values show mean of at least 3 independent experiments with 6 replicates each. Error bars indicate standard error.