Science - USA (2022-01-21)

Zhuet al.,Science 375 , eabg9765 (2022) 21 January 2022 7 of 11

2 3 4 5 6 7 8 9 10 11

Number of transcription factors (N)

101

10

2

10

3

Number of fixed points

= 0.8, = 20

Total stable fixed points

Robust stable fixed points

Limiting function of 2N

A Experimental MultiFate-3 design

A-only

B-only

C-only

A + B

A + C

B + C

A + B + C

0h 96h 144h

C Time-lapse imaging reveals MultiFate-3 septastability

37bs-37bs

inactive

complex

BCRbs-BCRbs

AD AD

AD

AD AD

AD

AD AD

ErBb2bs-ErbB2bs

AD

AD AD

AD AD AD AD

ABC

10x 10x 10x

High TMP, Image at 144h

D MultiFate can be expanded to generate more states

A-only B-only

A + C

A + B + C

C-only

A + B

B + C

Raw data + Gating Calculate cell fractions

Initial state

A-only

B-only

C-only

A + B

A + C

B + C

OFF

A + B + C

B MultiFate-3 cells generate up to 7 stable states

Day 0 Day 18

High TMP

Day 0 Day 18

Intermediate TMP

Day 0 Day 18

Low TMP

Unstable states - At least 10% cells escaped initial states

48h

Type II septastability Hexastability Tristability

Initial state

0

20

40

60

80

100

% of cells

OFF

(^10106)
(^105)
4
106
105
104
105
104
103
TF A (mC
herry
)
TF C (mTurquoise2)
TF B (m
Citrine)
0610 1066
105
0
105
10
Fig. 5. MultiFate architecture is expandable to include three and potentially
even more transcription factors.(A) The experimental MultiFate-3 design uses
three self-activation cassettes differing only in their fluorescent proteins and their
ZF DNA binding domains and binding sites. Each cassette expresses FKBP-ZF-
VP16-DHFR-IRES-FP-PEST, where ZF represents either BCRZFR39A, 37ZFR2AR11-
AR39AR67A, or ErbB2ZFR2AR39A and FP represents either mCherry, mCitrine, or
mTurquoise2 for A, B, and C, respectively. See table S2 for detailed construct
maps. (B) The MultiFate-3 line exhibited type II septastability, hexastability, and
tristability in three different TMP conditions. Top: State percentages in each octant
were quantified and plotted as eight colored circles ( 25 ). Bottom: High-TMP
condition = 100 nM AP1903 + 100 nM TMP; intermediate-TMP condition = 100 nM
AP1903 + 40 nM TMP; low-TMP condition = 100 nM AP1903 + 10 nM TMP. Except
for OFF-state cells, cells in different initial states were sorted from a mixed
population of cells in the high-TMP condition. Initial OFF cells came from cells in
regular CHO media without any inducers. Each plot represents the mean
percentage of three biological replicates. (C) Cells in each of the seven states were
stable during growth from single cells into colonies over 6 days under a time-lapse
microscope. We sorted cells and seeded an equal ratio of cells in seven states
using the same method as in Fig. 3D. Scale bars, 500mm for the wide-field image,
100 mm for zoomed-in images. (D) MultiFate is expandable. The number of
robust stable fixed points grows monotonically with the number of transcription
factor species (N) in the model. A robust stable fixed point is defined as a
stable fixed point that has fewer than 10% of cells escaping at the end of
stochastic simulations ( 25 ). The parameter set provided above the plot (with
Kd= 1 andn= 1.5) is the same nondimensionalized parameter set used in
MultiFate-2 and MultiFate-3 models under high protein stability.
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