Nescient helix-loop-helix 1 (Nhlh1) is a novel activating transcription factor 5 (ATF5) target gene in olfactory and vomeronasal sensory neurons in mice.

Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5-/-) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons.

Functional validation of epitope-tagged ATF5 knock-in mice generated by improved genome editing of oviductal nucleic acid delivery (i-GONAD).

Activating transcription factor 5 (ATF5) is a stress-responsive transcription factor that belongs to the cAMP response element-binding protein (CREB)/ATF family, and is essential for the differentiation and survival of sensory neurons in murine olfactory organs. However, the study of associated proteins and target genes for ATF5 has been hampered due to the limited availability of immunoprecipitation-grade ATF5 antibodies. To overcome this issue, we generated hemagglutinin (HA)-tag knock-in mice for ATF5 using CRISPR/Cas9-mediated genome editing with one-step electroporation in oviducts (i-GONAD). ATF5-HA fusion proteins were detected in the nuclei of immature and some mature olfactory and vomeronasal sensory neurons in the main olfactory epithelium and vomeronasal organ, respectively, as endogenous ATF5 proteins were expressed, and some ATF5-HA proteins were found to be phosphorylated. Chromatin immunoprecipitation (ChIP) experiments revealed that ATF5-HA bound to the CCAAT/enhancer-binding protein (C/EBP)-ATF response element site in the promotor region of receptor transporting protein 1 (Rtp1), a chaperone gene responsible for proper olfactory receptor expression. These knock-in mice may be used to examine the expression, localization, and protein-protein/-DNA interactions of endogenous ATF5 and, ultimately, the function of ATF5 in vivo.