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Design of Large-Scale Reporter Construct Arrays for Dynamic, Live Cell Systems Biology.

Joseph T Decker1, Matthew S Hall1, Beatriz Peñalver-Bernabé2

  • 1Department of Biomedical Engineering , University of Michigan , Ann Arbor , Michigan 48109 , United States.

ACS Synthetic Biology
|September 7, 2018
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This study developed novel transcription factor (TF) reporters to understand cell fate decisions. These reporters integrate TF binding with genetic activity, revealing key signaling hubs in response to TGFβ stimulation.

Keywords:
activity reportersynthetic promotertranscription factor

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Area of Science:

  • Systems biology
  • Molecular mechanisms
  • Cell fate decisions

Background:

  • Reporter constructs provide molecular insights into cellular processes.
  • CRISPR/Cas9 technology enables precise insertion of luciferase reporters for gene activity.
  • Understanding transcription factor (TF) binding is crucial for deciphering cellular responses.

Purpose of the Study:

  • To investigate design criteria for specific and responsive TF reporters.
  • To integrate TF binding activity with genetic reporter output.
  • To identify molecular mechanisms governing cell fate decisions.

Main Methods:

  • Designed and tested a library of 25 SMAD3 activity reporters with varying spacer sequences.
  • Utilized TF binding site prediction algorithms (BEEML, FIMO, DeepBind) to assess off-target binding.
  • Inferred TF activity networks and integrated them with large-scale genetic reporter measurements.

Main Results:

  • A spacer sequence was essential for quantifying activity changes post-TGFβ stimulation.
  • Off-target binding predictions correlated with reporter non-responsiveness, notably with p53 binding.
  • Integration of TF and genetic reporters identified major hubs involved in TGFβ signaling.

Conclusions:

  • Developed a systems-level algorithm for investigating cell signaling pathways.
  • Demonstrated the utility of integrated TF and genetic reporters for dissecting complex biological networks.
  • Provided insights into the molecular mechanisms controlling cell fate decisions through dynamic systems biology.