Science - USA (2022-01-21)

Zhuet al.,Science 375 , eabg9765 (2022) 21 January 2022 3 of 11

mCitrine- mCitrine+

WT R39A R2AR39AR2AR39AR67A

Mutations

1

10

20

30

40

Fold Activation

ErbB2ZF

ZF-AD

ZF-GCN4-AD

C TMP and AP1903 modulate autoregulation

0.0

0.2

0.4

0.6

0.8

1.0

mCit+ fraction, norm. to Dox group

FKBP-BCRZFR39A-VP48-DHFR self-activation

AP1903

TMP (nM)

• 
  

  0

  
  

  10000 0

  
  

+

1

+

10

+

100

+

10000

+

1000

+

A Engineered TFs exhibit dimer-dependent activity

D Competitive dimerization allows inhibition of positive autoregulation

ZF-AD

AD

Citrine

ZFbs-ZFbs

AD AD

X

TMP

Dox

mCitrine-PEST

ZFbs-ZFbs

FKBP-ZF-VP48-DHFRIRES

AP1903

AD AD AD

AD AD

B FKBP12F36V allows inducible dimerization

ZF-GCN4-AD

Citrine

ZFbs-ZFbs

AD AD

AP1903

FKBP-ZF-AD

Citrine

ZFbs-ZFbs

AD

AD AD

6x

2x

2x

2x

AD

Dox+ Dox+ (for norm.)

Dox+ AP1903+, TMP 10 μM

Dox+ AP1903+, TMP 1 nM

Dox+ no AP1903, TMP 10 μM

0 0.1 1 10 100

AP1903 (nM)

1

5

10

15

Fold Activation

DHFR degron

Zinc finger DNA-binding domains FKBP12F36V homodimerization domain

ADTranscriptional activation domain

Monomeric or dimeric GCN4 domain

AP1903 dimerizer TMP stabilizing drug

AD AD

Monoclonal

stable line 1

AD

inactive

complex

active

complex

AD AD

perturbation

ZFbs-ZFbs

IRESmCitrine-PEST

6x

ZF-GCN4-VP48-DHFR

AD

AD AD

Monoclonal

stable line 2

inactive

complex

active

complex

ZFbs-ZFbs

IRESmCitrine-PEST

6x

FKBP-ZF-VP48-DHFR

AD AD

Perturbation

None AD AD

AD

0.0

0.2

0.4

0.6

0.8

1.0

mCitrine+ fraction

Monoclonal stable line 1

None AD

AD

Perturbation

AD

0.0

0.2

0.4

0.6

0.8

1.0

mCitrine+ fraction

Monoclonal stable line 2

perturbation

AD AD

AD

or or

AD AD

AD

or or

Fig. 2. Engineered transcription factors enable homodimer-dependent auto-
regulation and heterodimerization-based inhibition.(A) ZF transcription factors
enable homodimer-dependent activation. Left: Design of test constructs in which
ErbB2ZF ( 31 ) (red circle), fused to VP48 (AD) and in some cases GCN4 (blue squiggle)
domains, binds to target sites (red pads) to activate Citrine expression. Activators were
expressed from a constitutive CAG promoter ( 52 ). Right: R-to-A mutations in ZF-
modulated reporter activation by ZF-GCN4-AD and ZF-AD. The R2AR39A variant was
selected because of high ZF-GCN4-AD activation and minimal ZF-AD activation. Fold
activation is defined in fig. S4A. WT, wild-type variant. (B) Left: Design of test constructs in
which FKBP12F36V (FKBP, light cyan partial box), fused to BCRZFR39A (red circle) and
VP48 (AD), binds to target sites (red pads) to activate Citrine expression in the presence of
AP1903 (cyan circle). Right: This design allows dose-dependent control of activation by
AP1903. (C) Transcription factor self-activation can be controlled by TMP and AP1903.

Left: Design of the controllable self-activation circuit. IRES, internal ribosome entry site;

PEST, constitutive degradation tag ( 53 ). Center: Stable polyclonal cells showed bimodal

mCitrine distribution upon circuit activation. An empirical threshold at mCitrine = 10^4

separates the distribution into two fractions, and the normalized mCitrine+fraction was

used to quantify the self-activation strength ( 25 ). Right: Colored arrows indicate data

from the center panel. AP1903+samples had 100 nM AP1903. (D) Self-activation was

inhibited by proteins with a different ZF and matching dimerization domains. Two

monoclonal stable lines could spontaneously self-activate in media containing AP1903

and TMP (fig. S5B). Each perturbation construct is introduced by stable integration ( 25 ).

The integrated construct in the“None”group did not express any perturbation protein.

Red circle denotes 42ZFR2AR39AR67A; green circle denotes BCRZFR39A. In all

panels, each dot represents one biological replicate, and each red line or bar indicates the

mean of replicates. Lists of constructs and cell lines are in tables S2 and S3.

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