{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:51:20Z","timestamp":1782280280115,"version":"3.54.5"},"posted":{"date-parts":[[2025,8,29]]},"group-title":"Genetics","reference-count":60,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T00:00:00Z","timestamp":1756425600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,23]]},"abstract":"<jats:sec>\n                  <jats:title>BACKGROUND<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS<\/jats:title>\n                  <jats:p>\n                    We generated multiple distinct CM-specific (\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(e2\/3\/pA)<\/jats:sup>\n                    <\/jats:italic>\n                    and\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(e2)<\/jats:sup>\n                    <\/jats:italic>\n                    ) and global\n                    <jats:italic>Atf4<\/jats:italic>\n                    knockout (\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>7del\/7del<\/jats:sup>\n                    <\/jats:italic>\n                    and\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>1ins\/1ins<\/jats:sup>\n                    <\/jats:italic>\n                    ) mouse models targeting different\n                    <jats:italic>Atf4<\/jats:italic>\n                    regions, as well as cardiomyocyte-specific deletion of\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    to study cardiac phenotypes. Detailed morphological and molecular analyses were performed.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS<\/jats:title>\n                  <jats:p>\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(<\/jats:sup>\n                    <\/jats:italic>\n                    <jats:sup>\n                      e2\/3\n                      <jats:italic>\/pA)<\/jats:italic>\n                    <\/jats:sup>\n                    (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\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    downregulation, a potent suppressor of p53. Further investigation revealed that deleting the polyA signal of\n                    <jats:italic>Atf4<\/jats:italic>\n                    in\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(e2\/3\/pA)<\/jats:sup>\n                    <\/jats:italic>\n                    mice led to transcriptional readthrough, resulting in the formation of an\n                    <jats:italic>Atf4<\/jats:italic>\n                    -\n                    <jats:italic>Cacna1i<\/jats:italic>\n                    fusion transcript and\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    downregulation. To avoid readthrough while abolishing ATF4 function, we introduced small indels into exon 3 of\n                    <jats:italic>Atf4<\/jats:italic>\n                    in mice (\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>7del\/7del<\/jats:sup>\n                    <\/jats:italic>\n                    and\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>1ins\/1ins<\/jats:sup>\n                    <\/jats:italic>\n                    ), which showed normal\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    expression and cardiac morphology. Importantly, CM-specific deletion of\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    recapitulated the cardiac defects and transcriptional change seen in\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(e<\/jats:sup>\n                    <\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    <jats:italic>\n                      <jats:sup>\/3\/pA)<\/jats:sup>\n                    <\/jats:italic>\n                    mice.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS<\/jats:title>\n                  <jats:p>\n                    We found that the downregulation of\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    , not loss of ATF4 function, underlying the cardiac phenotypes in\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(e2\/3\/pA)<\/jats:sup>\n                    <\/jats:italic>\n                    mice. The reduced expression of\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    in\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>cKO(e2\/3\/pA)<\/jats:sup>\n                    <\/jats:italic>\n                    mice is likely due to the unintentional deletion of\n                    <jats:italic>Atf4<\/jats:italic>\n                    polyA signal and subsequent transcriptional readthrough, underscoring the essential role of RPS19BP1, not ATF4, in cardiac development. Consistent\n                    <jats:italic>Rps19bp1<\/jats:italic>\n                    downregulation has been observed in other tissue-specific\n                    <jats:italic>Atf4<\/jats:italic>\n                    knockout models utilizing the\n                    <jats:italic>\n                      Atf4\n                      <jats:sup>fl(e2\/3\/pA)<\/jats:sup>\n                    <\/jats:italic>\n                    allele, suggesting that previously reported\n                    <jats:italic>Atf4<\/jats:italic>\n                    KO phenotypes may result from\n                    <jats:italic>Atf4<\/jats:italic>\n                    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.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>TRANSLATIONAL PERSPECTIVE<\/jats:title>\n                  <jats:p>Our findings clarify ATF4\u2019s role in heart development by showing that cardiac defects in cardiomyocyte-specific ATF4 knockout mice\u2014using a widely employed floxed ATF4 line\u2014result from unintended downregulation of RPS19BP1 caused by transcriptional readthrough. This shifts the focus from ATF4 to RPS19BP1, a key regulator of p53 activity, as a potential driver of cardiac developmental abnormalities. Clinically, these insights caution against misinterpretation of genetic knockout models and highlight RPS19BP1 as a promising target for congenital heart disease and related cardiac dysfunctions, with potential implications for future therapies.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.08.26.672495","type":"posted-content","created":{"date-parts":[[2025,8,30]],"date-time":"2025-08-30T00:00:15Z","timestamp":1756512015000},"source":"Crossref","is-referenced-by-count":0,"title":["Transcriptional Readthrough at\n                  <i>Atf4<\/i>\n                  Locus Suppresses\n                  <i>Rps19bp1<\/i>\n                  and Impairs Heart 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