Transcription Factor ZBTB6 Activates the Transcription and Function of NORHA in Sow Granulosa Cells

This study was aimed to characterize the distal core promoter of the porcine lncRNA NORHA gene, and explore the mechanism by which the transcription factor (TF) ZBTB6 activated its transcription and function, so as to provide a basis for analyzing the transcriptional regulatory network of NORHA.

The sequence of the distal core promoter of the NORHA gene in Yorkshire pigs was obtained by PCR amplification and sequencing. Bioinformatic method was used to predict the TF-binding sites (TFBSs) and to characterize the ZBTB6 encoding sequence. A ZBTB6 overexpression vector was constructed by restriction enzymatic digestion, and its overexpression efficiency in sow granulosa cells (sGCs) was verified by qPCR and western blot. The effects of ZBTB6 on the activity of the distal core promoter and transcription of NORHA in sGCs were determined by luciferase assay and qPCR. The binding of ZBTB6 to the ZBTB6-binding site (ZBS) in the distal core promoter in sGCs was detected by chromatin immunoprecipitation (ChIP) and DNA sequencing. The concentrations of progesterone (P4) and estradiol (E2) in follicular fluid were measured by ELISA. ZBTB6 and NORHA levels in follicles, and BCL2 and BAX levels in ZBTB6-overexpressing sGCs were detected by qPCR.

TFBSs of 255 TFs, including ZBTB6, were predicted in 526-bp of the distal core promoter of Yorkshire NORHA gene. The overexpression vector successfully achieved the overexpression of ZBTB6 levels in sGCs. The transcriptional levels of NORHA in sGCs were markedly upregulated in ZBTB6-overexpressing sGCs. Correlation analysis revealed that ZBTB6 and NORHA levels in follicles were markedly positively correlated. A ZBS motif was discovered at -2276/-2264 nt in the distal core promoter. Luciferase assay revealed that ZBTB6 activates the activity of the distal core promoter via the ZBS motif. ChIP and sequencing confirmed that ZBTB6 directly bound to the ZBS motif in the distal core promoter of the NORHA gene in sGCs. The ZBTB6 gene in pigs and other mammalian species was evolutionally conserved and all contained a conserved DNA-binding domain. ZBTB6 was markedly upregulated during sow follicular atresia and showed a marked positive correlation with the P4/E2 ratio, a marker for follicular atresia. Co-transfection experiments showed that overexpression of ZBTB6 markedly downregulated the anti-apoptotic gene BCL2 levels, upregulated the pro-apoptotic gene BAX levels, and downregulated the BCL2/BAX ratio, an apoptotic marker, whereas knockdown of NORHA reversed this.

ZBTB6 was a transcription activator of the NORHA gene in sGCs, which accelerated sGC apoptosis and follicular atresia by directly activating NORHA transcription.