bioRxiv [Preprint]. 2025 Aug 29:2025.08.26.672495. doi: 10.1101/2025.08.26.672495.
ABSTRACT
BACKGROUND: Activating Transcription Factor 4 (ATF4) functions as a transcriptional regulator in various cell types and tissues under both physiological and pathological conditions. While previous studies have linked ATF4 activation with promoting cardiomyocyte (CM) death in dilated cardiomyopathy (DCM), atrial fibrillation, and heart failure, its role in developing CMs remains unexplored.
METHODS: We generated multiple distinct CM-specific ( Atf4 cKO(e2/3/pA) , Atf4 cKO(e2) and Atf4 cKO(e2/3/nopA) ) and global Atf4 knockout ( Atf4 7del/7del and Atf4 1ins/1ins ) mouse models targeting different Atf4 regions, as well as cardiomyocyte-specific deletion of Rps19bp1 to study cardiac phenotypes. Detailed morphological and molecular analyses were performed.
RESULTS: Atf4 cKO(e2/3/pA) (targeting exon 2-3 including the polyadenylation signal (polyA)) mice exhibited severe cardiac defects and died before E17.5, likely due to ectopic activation of p53 signaling pathway resulting from Rps19bp1 downregulation, a potent suppressor of p53. Further investigation revealed that deleting the polyA signal of Atf4 in Atf4 cKO(e2/3/pA) mice led to transcriptional readthrough, resulting in the formation of an Atf4 - Cacna1i fusion transcript and Rps19bp1 downregulation. To avoid readthrough while abolishing ATF4 function, we introduced small indels into exon 3 of Atf4 in mice ( Atf4 7del/7del and Atf4 1ins/1ins ), which showed normal Rps19bp1 expression and cardiac morphology. Furthermore, CM-specific deletion of exons 2-3 while retaining the polyA signal ( Atf4 cKO(e2/3/nopA) ) mice had no effect on cardiac development. Importantly, CM-specific deletion of Rps19bp1 recapitulated the cardiac defects and transcriptional change seen in Atf4 cKO(e2/3/pA) mice.
CONCLUSIONS: We found that the downregulation of Rps19bp1 , not loss of ATF4 function, underlying the cardiac phenotypes in Atf4 cKO(e2/3/pA) mice. The reduced expression of Rps19bp1 in Atf4 cKO(e2/3/pA) mice is likely due to the unintentional deletion of Atf4 polyA signal and subsequent transcriptional readthrough, underscoring the essential role of RPS19BP1, not ATF4, in cardiac development. Consistent Rps19bp1 downregulation has been observed in other tissue-specific Atf4 knockout models utilizing the Atf4 fl(e2/3/pA) allele, suggesting that previously reported Atf4 KO phenotypes may result from Atf4 transcriptional readthrough effects. These findings reveal a locus-dependent transcriptional interference mechanism and emphasize the importance of avoiding confounding cis effects in genetically engineered models.
PMID:40909608 | PMC:PMC12407945 | DOI:10.1101/2025.08.26.672495