RESUMEN
Retinoic acid-related orphan receptor γ (RORγ) maintains the circadian rhythms of its downstream genes. However, the mechanism behind the transcriptional activation of RORγ itself remains unclear. Here, we demonstrate that transcription of RORγ is activated by heterogeneous nuclear ribonucleoprotein K (hnRNP K) via the poly(C) motif within its proximal promoter. Interestingly, we confirmed the binding of endogenous hnRNP K within RORγ1 and RORγ2 promoter along with the recruitment of RNA polymerase 2 through chromatin immunoprecipitation (ChIP). Furthermore, an assay for transposase accessible chromatin (ATAC)-qPCR showed that hnRNP K induced higher chromatin accessibility within the RORγ1 and RORγ2 promoter. Then we found that the knockdown of hnRNP K lowers RORγ mRNA oscillation amplitude in both RORγ and RORγ-dependent metabolic genes. Moreover, we demonstrated that time-dependent extracellular signal-regulated kinase (ERK) activation controls mRNA oscillation of RORγ and RORγ-dependent metabolic genes through hnRNP K. Taken together, our results provide new insight into the regulation of RORγ by hnRNP K as a transcriptional activator, along with its physiological significance in metabolism.
Asunto(s)
Cromatina/metabolismo , Ritmo Circadiano/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Animales , Inmunoprecipitación de Cromatina/métodos , Ritmo Circadiano/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ratones , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Although proliferation of keratinocytes, a major type of skin cells, is a key factor in maintaining the function of skin, their ability to proliferate tends to diminish with age. To solve such a problem, researchers in medical and skin cosmetic fields have tried to utilize epidermal growth factor (EGF), but achieved limited success. Therefore, a small natural compound that can mimic the activity of EGF is highly desired in both medical and cosmetic fields. Here, using the modified biosensor system, we observed that natural small-compound isoprocurcumenol, which is a terpenoid molecule derived from turmeric, can activate EGFR signaling. It increased the phosphorylation of ERK and AKT, and upregulated the expression of genes related to cell growth and proliferation, such as c-myc, c-jun, c-fos, and egr-1. In addition, isoprocurcumenol induced the proliferation of keratinocytes in both physical and UVB-induced cellular damage, indicative of its function in skin regeneration. These findings reveal that EGF-like isoprocurcumenol promotes the proliferation of keratinocytes and further suggest its potential as an ingredient for medical and cosmetics use.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Regeneración/efectos de los fármacos , Sesquiterpenos/farmacología , Activación Transcripcional/efectos de los fármacos , Línea Celular , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Queratinocitos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Sesquiterpenos/química , Transducción de Señal/efectos de los fármacos , Piel/crecimiento & desarrollo , Piel/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Circadian gene expression is defined by the gene-specific phase and amplitude of daily oscillations in mRNA and protein levels. D site-binding protein mRNA (Dbp mRNA) shows high-amplitude oscillation; however, the underlying mechanism remains elusive. Here, we demonstrate that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator that activates Dbp transcription via the poly(C) motif within its proximal promoter. Biochemical analyses identified hnRNP K as a specific protein that directly associates with the poly(C) motif in vitro Interestingly, we further confirmed the rhythmic binding of endogenous hnRNP K within the Dbp promoter through chromatin immunoprecipitation as well as the cycling expression of hnRNP K. Finally, knockdown of hnRNP K decreased mRNA oscillation in both Dbp and Dbp-dependent clock genes. Taken together, our results show rhythmic protein expression of hnRNP K and provide new insights into its function as a transcriptional amplifier of Dbp.
Asunto(s)
Ritmo Circadiano/genética , Proteínas de Unión al ADN/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Células 3T3 , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Poli C/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
The D site-binding protein (Dbp) supports the rhythmic transcription of downstream genes, in part by displaying high-amplitude cycling of its own transcripts compared to other circadian-clock genes. However, the underlying mechanism remains elusive. Here, we demonstrated that the poly(C) motif within the Dbp proximal promoter, in addition to an E-box element, provoked transcriptional activation. Furthermore, we generated a cell line with poly(C) deleted to demonstrate the endogenous effect of the poly(C) motif within the Dbp promoter. We investigated whether RNA polymerase 2 (Pol2) recruitment on the Dbp promoter was decreased in the cell line with poly(C) deleted. Next, assay for transposase-accessible chromatin (ATAC)-quantitative PCR (qPCR) showed that the poly(C) motif induced greater chromatin accessibility within the region of the Dbp promoter. Finally, we determined that the oscillation amplitude of endogenous Dbp mRNA of the cell line with poly(C) deleted was decreased, which affected the oscillation of other clock genes that are controlled by Dbp Taken together, our results provide new insights into the function of the poly(C) motif as a novel cis-acting element of Dbp, along with its significance in the regulation of circadian rhythms.