Molecular and Cellular Biochemistry

, Volume 440, Issue 1–2, pp 65–75 | Cite as

Functional validation of ATF4 and GADD34 in Neuro2a cells by CRISPR/Cas9-mediated genome editing

  • Kentaro Oh-hashi
  • Naoki Sugiura
  • Fumimasa Amaya
  • Ken-ichi Isobe
  • Yoko Hirata
Article

Abstract

Activating transcription factor 4 (ATF4), which is ubiquitously expressed, plays a crucial role in regulating various stress-responsive genes under pathophysiological conditions. Further, growth arrest and DNA damage-inducible gene 34 (GADD34), a downstream target of ATF4, has been reported to negatively regulate ATF4 expression. To understand the relationship between intrinsic ATF4 and GADD34 under resting and ER stress conditions, we used a novel gene editing approach, CRISPR/Cas9, to integrate antibiotic-resistant genes into the target genes, ATF4 and GADD34. First, we manipulated the ATF4 gene in the mouse neuroblastoma cell line, Neuro2a, and compared the ER stress responses between parental and ATF4-edited Neuro2a cells. Next, we established Neuro2a cells with edited GADD34 and ATF4/GADD34 genes and found that ATF4 acts as a proapoptotic factor, but GADD34 depletion did not attenuate the expression of cleaved caspase-3 induced by tunicamycin treatment. These findings provide new insights into the ATF4 signaling cascades. Additionally, the rapid establishment of cells lacking multiple genes using this CRISPR/Cas9 system will be a powerful tool for exploring various cellular issues under pathophysiological conditions.

Keywords

ATF4 CRIPSR/Cas9 ER stress GADD34 

Abbreviations

ATF4

Activating transcription factor 4

ATF6

Activating transcription factor 6

eIF2α

Eukaryotic translation initiation factor 2α

ER

Endoplasmic reticulum

GADD34

Growth arrest and DNA damage-inducible gene 34

GADD153

Growth arrest and DNA damage-inducible gene 153

G3PDH

Glyceraldehyde 3-phosphate dehydrogenase

GRP78

78-kDa glucose-regulated protein

Herp

Homocysteine-induced endoplasmic reticulum protein

IRE1

Inositol-requiring enzyme-1

PERK

PKR-like endoplasmic reticulum kinase

RT-PCR

Reverse transcription polymerase chain reaction

Notes

Acknowledgements

We thank Dr. George Church for providing the hCas9. This work is, in part, supported by Grant-in-Aid for Challenging Exploratory Research (No. 17K19901 to K.O.), Grant-in-Aid for Scientific Research (B) (No. 26670692, to F.A., and No. 16H05280, to K.I.), and the OGAWA Science and Technology Foundation (to K.O.).

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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Kentaro Oh-hashi
    • 1
    • 2
  • Naoki Sugiura
    • 2
  • Fumimasa Amaya
    • 3
  • Ken-ichi Isobe
    • 4
  • Yoko Hirata
    • 1
    • 2
  1. 1.United Graduate School of Drug Discovery and Medical Information SciencesGifu UniversityGifuJapan
  2. 2.Department of Chemistry and Biomolecular Science, Faculty of EngineeringGifu UniversityGifuJapan
  3. 3.Department of AnesthesiologyKyoto Prefectural University of MedicineKyotoJapan
  4. 4.Nagoya Wemen UniversityNagoyaJapan

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