CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells
Bibliographic record
Abstract
The objective of this study was to determine the role of GCN2 in the response to AA deprivation of primary bovine mammary epithelial cells (BMEC). Cells were isolated from the mammary tissue of 2 lactating Holstein cows by enzymatic digestion, expanded, and induced to differentiate for 5 to 7 d. Relative mRNA expression was measured by real-time quantitative PCR. Protein abundance and site-specific phosphorylation were measured by immunoblotting. Knockout of GCN2 in BMEC was accomplished by lentiviral delivery of a targeted single guide RNA and endonuclease Cas9. To investigate the role of GCN2, we treated lactogenic differentiated BMEC with either culture medium lacking Arg, Leu, and Lys combined or lacking only one of the 3 AA of interest, in comparison to a control with a full complement of AA. Activation of GCN2 was inferred by the phosphorylation status of its downstream target eIF2α Ser51. We found that GCN2 was activated by both the deprivation of Arg, Leu, and Lys combined and of Arg alone, as shown by a 2.73- and 2.82-fold increase in phosphorylated eIF2α Ser51 after 1 h of deprivation, respectively. In addition, activation of GCN2 as measured by increased phosphorylation of eIF2α Ser51 during the deprivation of Arg, Leu, and Lys combined and of Arg alone was sustained for up to 8 h of deprivation. Phosphorylated eIF2α selectively upregulates translation of transcription factor ATF4, among others, during AA deprivation, which then targets genes necessary for restoring AA homeostasis. Therefore, we investigated the expression of ATF4 transcriptional targets, AA enzyme ASNS and AA transporters SLC7A1 and SLC38A2. We found that ASNS was upregulated in response to combined AA deprivation and by Arg deprivation alone by 3.6- and 4.51-fold, respectively, at 24 h of treatment. We found that SLC7A1 was upregulated in response to combined AA deprivation and deprivation of Arg alone by 2.0- and 2.36-fold, respectively, at 8 h of treatment. To establish the role of GCN2 (encoded by EIF2AK4) in the response to AA deprivation, we ablated GCN2 in BMEC using clustered regularly interspaced short palindromic repeats-Cas9. We showed that BMEC transduced with single guide RNA targeting EIF2AK4 were not as responsive to combined AA deprivation, compared with BMEC transduced with nontargeting single guide RNA. Taken together, our results demonstrate a critical role for GCN2 in sensing AA deprivation in BMEC.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".