Transcriptional Readthrough at Locus Suppresses and Impairs Heart Development.

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 ( , and ) and global knockout ( and ) mouse models targeting different regions, as well as cardiomyocyte-specific deletion of to study cardiac phenotypes. Detailed morphological and molecular analyses were performed.

RESULTS

(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 downregulation, a potent suppressor of p53. Further investigation revealed that deleting the polyA signal of in mice led to transcriptional readthrough, resulting in the formation of an - fusion transcript and downregulation. To avoid readthrough while abolishing ATF4 function, we introduced small indels into exon 3 of in mice ( and ), which showed normal expression and cardiac morphology. Furthermore, CM-specific deletion of exons 2-3 while retaining the polyA signal ( ) mice had no effect on cardiac development. Importantly, CM-specific deletion of recapitulated the cardiac defects and transcriptional change seen in mice.

CONCLUSIONS

We found that the downregulation of , not loss of ATF4 function, underlying the cardiac phenotypes in mice. The reduced expression of in mice is likely due to the unintentional deletion of polyA signal and subsequent transcriptional readthrough, underscoring the essential role of RPS19BP1, not ATF4, in cardiac development. Consistent downregulation has been observed in other tissue-specific knockout models utilizing the allele, suggesting that previously reported KO phenotypes may result from 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.