Science - USA (2022-01-21)

Zhuet al.,Science 375 , eabg9765 (2022) 21 January 2022 4 of 11

B

D Time-lapse imaging reveals MultiFate-2.3 tristability

High TMP, Image at 119h 0h 119h

A-only

B-only

A + B

A Experimental MultiFate-2 design

37bs-37bs

inactive

complex

BCRbs-BCRbs

AD AD

AD

AD AD

AD

AD AD

AB

6x or 10x 6x or 10x

Dox

4-OHT

or Dox

C MultiFate-2 cell lines exhibit bistability and tristability

A-only

B-only A + B

OFF

OFF A-only

B-only A+B

OFF A-only

B-only A+B

+ AP1903

+ TMP

3 days

A-only

B-only A + B

OFF

+ AP1903

+ TMP

3 days

Unstable states - At least 10% cells escaped initial states

MultiFate-2.3

High TMP

Day 0 Day 18

OFF

A-only

A + B

B-only

Initial state

Type II tristability

Low TMP

Day 0 Day 18

Bistability

MultiFate-2.2

Day 0 Day 18

Low TMP

Day 0 Day 18

High TMP

Type II tristability Bistability

OFF

A-only

B-only

A + B

Initial state

MultiFate-2.1

Day 0 Day 18

Low TMP

Day 0 Day 18

High TMP

Type II tristability Bistability

OFF

A-only

B-only

A + B

Initial state

0

20

40

60

80

100

% of cells

60h

OFF A-only

A+B

TF A Expression

(mCherry) (a.u.)

102

103

104

105

106

102103104105106

TF B Expression (mCitrine) (a.u.)

FACS

Mixed at equal ratio

TF A Expression

(mCherry) (a.u.)

TF B Expression (mCitrine) (a.u.)^103

104

105

106

102 103 104 105 106

TF A Expression

(mCherry) (a.u.)

103

104

105

106

102 103 104 105 106

B-only

Fig. 3. MultiFate-2 generates multiple stable states.(A) The experimental
MultiFate-2 design uses two 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 or 37ZFR2AR11AR39AR67A and FP represents either mCherry or
mCitrine, for A and B, respectively. Detailed construct maps and differences
amongMultiFate-2linesareshownintablesS2andS3.(B) MultiFate-2.1 cells
spontaneously activate A, B, or both cassettes upon addition of 100 nM AP1903
and 10mM TMP. Cell percentages in OFF, A-only, B-only, and A+B states were
quantified and plotted as a square with four colored circles ( 31 ); a.u., arbitrary
units. (C) Three MultiFate-2 lines all exhibited type II tristability in the high-TMP
condition and bistability in the low-TMP condition. In all conditions, we added

100 nM AP1903. Exact concentrations of TMP are shown in figs. S9 to S11. Unstable

states, defined by states having more than 10% cells escaping their initial states

after 18 days, were marked in pink rectangles. Each square represents the mean

fractions of three biological replicates. Initial A-only, B-only, and A+B cells were

sorted from a population of cells in different states, while initial OFF cells came from

cells in regular CHO media without any inducers. (D) A-only, B-only, and A+B states

were each stable during growth from single MultiFate-2.3 cells into colonies over

5 days (119 hours) under a time-lapse microscope. Left: We first sorted mixed

MultiFate-2.3 cell populations to separate cells in three different states. Then

we seeded cells in these three states at equal ratio in the same well and performed

time-lapse imaging ( 25 ). Scale bars, 500mm for the wide-field image, 100mm for

zoomed-in images.“High TMP”= 100 nM AP1903 + 10mM TMP.

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