Transcriptional readthrough at Atf4 locus suppresses Rps19bp1 and impairs heart development

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AIMS: 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 AND RESULTS: We generated multiple distinct CM-specific (Atf4cKO(e2/3/pA) and Atf4cKO(e2)) and global Atf4 knockout (KO; Atf47del/7del and Atf41ins/1ins) mouse models targeting different Atf4 regions, as well as CM-specific deletion of Rps19bp1 to study cardiac phenotypes. Detailed morphological and molecular analyses were performed. Atf4cKO(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 the p53 signaling pathway resulting from Rps19bp1 downregulation, a potent suppressor of p53. Further investigation revealed that deleting the polyA signal of Atf4 in Atf4cKO(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 (Atf47del/7del and Atf41ins/1ins), which showed normal Rps19bp1 expression and cardiac morphology. Importantly, CM-specific deletion of Rps19bp1 recapitulated the cardiac defects and transcriptional change seen in Atf4cKO(e2/3/pA) mice. CONCLUSION: We found that the downregulation of Rps19bp1, not the loss of ATF4 function, underlies the cardiac phenotypes in Atf4cKO(e2/3/pA) mice. The reduced expression of Rps19bp1 in Atf4cKO(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 KO models utilizing the Atf4fl(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.

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