Supplementary Information

Optimized second generation CRY2/CIB dimerizers and photoactivatable Cre recombinase

1Amir Taslimi, 2Brian Zoltowski, 1Jose G. Miranda, 1Gopal Pathak, 3Robert M. Hughes, 1Chandra L. Tucker

1Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045 2Department of Chemistry, Southern Methodist University, Dallas, TX 75275 3Department of Biology, Duke University, Durham, NC  

1

Nature Chemical Biology: doi:10.1038/nchembio.2063

BD-CRY2PHR+ AD-CIB1

BD-CRY2FL+ AD-CIB1

BD-CRY2(515)+ AD-CIB1

BD-CRY2(535)+ AD-CIB1

Dark Light

Supplementary Figure 1. Interaction of CRY2 truncations with CIB1 visualized by yeast two-hybrid. Shown are AH109/Y187 yeast expressing indicated Gal4BD-CRY2 truncation constructs tested for interaction with GalAD-CIB1. Positive interaction induces His3 reporter activity, allowing growth on SD -Trp/-Leu/-His +3mM 3AT plates. Dark samples were kept in the dark, while light samples were subjected to blue light pulses (1s pulse every 3 min, 461 nm, 5.8 mW/cm2) for 52 hrs.Growth assays were repeated twice with similar results.

Supplementary Results

2

Nature Chemical Biology: doi:10.1038/nchembio.2063

BD-CRY2PHR+ pGADT7rec(empty vector)

BD-CRY2PHR+ AD-CRY2PHR

Light Dark

BD-CRY(515)+ AD-CRY(515)

BD-CRY(535)+ AD-CRY(535)

Supplementary Figure 2. CRY2 truncation constructs tested for self-interaction. Shown are AH109/Y187 yeast expressing indicated CRY2 truncation constructs. Positiveinteraction induces His3 reporter activity, allowing growth on SD –Trp/-Leu/-His +3mM 3ATplates. Dark samples were kept in the dark, while light samples were subjected to blue light pulses (1s pulse every 3 min, 461 nm, 5.8 mW/cm2) for 48 hrs. CRY2FL and CRY2(535) show a substantial reduction in dark self-interaction, compared with CRY2PHR and CRY2(515). Experiments were repeated twice with similar results

BD-CRY2FL+ AD-CRY2FL

Nature Chemical Biology: doi:10.1038/nchembio.2063

0

25

50

75

100

0.5 1.0 1.5

Pre lightPost light

Distance (µm)

Fluo

resc

ence

inte

nsity

(A.U

.)

Pre light Post light

2.00.5 1.0 1.5

Pre lightPost light

0

25

50

75

100

Fluo

resc

ence

inte

nsity

(A.U

.)

Distance (µm)

CIB28-pmEFPCRY2PHR-mCh

a

b

Light Dark

BD-CRY2FL+ AD-CIB28

BD-CRY2FL+ AD-CIBN

BD-CRY2FL+ pGADT7rec(empty vec)

Supplementary Figure 3. Functional tests of interaction of CRY2PHR with CIB28. (a) Plasma membrane recruitment of CRY2PHR-mCherry to CIB28 following a pulse of light. Shownare images of HEK293T cells expressing CRY2PHR-mCherry and membrane-anchored CIB28-pmGFP (containing a CaaX motif at the C-terminus of EGFP) before and 30 s post blue light exposure. CRY2PHR-mCherry is weakly recruited to the plasma membrane by CIB28. Graphs at right show quantification of fluorescence intensity at membrane at indicated areas. Scale bars, 5 µm.(b) Yeast two-hybrid interaction analysis of AH109/Y187 yeast expressing indicated constructs.Yeast expressing Gal4BD-CRY2FL and Gal4AD-CIB28 show substantial growth in dark, compared with Gal4BD-CRY2FL + Gal4AD-CIBN. Yeast were grown in dark or subjected to blue light pulses (1s pulse every 3 min, 461 nm, 5.8 mW/cm2) for 48 hrs. Experiments were repeated three times with similar results.

4

Nature Chemical Biology: doi:10.1038/nchembio.2063

0

25

50

75

100

origin

al(10

4/106

)

60/61

66/67

101/1

02

109/1

10

279/2

80

Dark Light

% R

ecom

bina

tion

Cre fragment split site

Supplementary Figure 4. Functional testing of different Cre recombinase split sites. N- and C-terminal fragments of Cre recombinase were generated at indicated split sites, and fused to CRY2 or CIB1, respectively. HEK293 cells were transfected with split Cre variants anda loxP-STOP-loxP-EGFP reporter, incubated for 24 hrs in dark, then exposed to 24 hrs blue lightpulses (5.8 mW/cm2, 461 nm, 1s pulse every 3 min) before quantification of Cre reporter activityby manual cell count. Data shows average of two independent experiments (triplicate replicates).

Supplementary Figure 5. Activity of second generation PA-Cre constructs with extended illumination. HEK293 cells transfected with a loxP-STOP-loxP-EGFP reporter, CRY2-L348F-CreN, and CIB1-CreC(N1) were incubated in the dark, then kept in dark or exposed to 24 hrs of light treatments (2s pulse every 3 min, 461 nm, 5.8 mW/cm2) beginning 24 hrs after transfection. Cre reporter activity was assayed 48 hrs after transfection by manual count. Data represent mean values ± s.d. (n=3) from one experiment, and experiments were repeated two times with similar results.

% R

ecom

bina

tion

0

20

40

60

80

WT L348F reporter

Dark Light

5

Nature Chemical Biology: doi:10.1038/nchembio.2063

ß-ga

lact

osid

ase

activ

ity(n

orm

aliz

ed, A

.U.)

Supplementary Figure 7. Comparison of wild-type and L348F CRY2 with a LexA-VP16 transcriptionalsystem. W303-1A cells were coexpressed with wild-type or L348F LexA-CRY2(535), VP16-CIB1, and a pSH18-34 transcriptional reporter. Shown is β-galactosidase reporter activity assayed in cells kept in dark, or 2 hr after treatment with a single flash of blue light (2s pulse, 461 nm, 5.8 mW/cm2). Graph shows normalized mean values from two independent experiments (n=3 replicates each).

1

10

100

1000

10000

wt L348F

Dark Light

Supplementary Video 1. Recruitment and dissociation of CRY2PHR-mCh with CIB81-pmEGFP. Shown are timelapse images of HEK293T cells expressing CRY2PHR-mCh and membrane localized CIB81-pmEGFP (containing a CaaX motif at C-terminus of EGFP) pre and post exposure to a single pulse of 488nm light (indicated by white dot on image stack). A time counter (mins) is indicated at top left.

Other Supplementary Information

Supplementary Table 1. Sequences of constructs used in studies (Excel spreadsheet).

Supplementary Figure 6. Activity of tagged and truncated versions of second generation PA-Creconstructs. HEK293 cells were cotransfected with a loxP-STOP-loxP-EGFP reporter and indicated CreN and CreC constructs and incubated in dark for 24 hrs. Cells were either kept in dark or exposed to a single 4 s pulse of blue light, then incubated in dark an additional 24 hrs before manual reporter quantification. Graph shows normalized mean values ± s.d. (n = 6 for FL/N1, n=3 for others) for three independent experiments. Data was normalized to results with CRY2-L348F-CreN + CIB1-CreC(N1)for comparison.

50

100 Light

Dark

CRY2-L348F-CreN:CIB1-CreC:

FL FLIRES CIB1-CreC-

2xHA-IRES

535N1 N1

FL Reporter onlymyc-FL

N1

150

Com

para

tive

Cre

reco

mbi

natio

n (n

orm

aliz

ed)

Nature Chemical Biology: doi:10.1038/nchembio.2063