Zhx2 and the Regulation of AFP Anti‐sense RNAs in Mouse Liver

Although the genomes of eukaryotes are mostly transcribed, only a very small percentage of the transcripts code for proteins. The majority of the transcripts do not have known functions. In recent years, growing evidence has suggested that these non‐coding transcripts may play important roles in gene regulation. In the past two years, our lab has identified and characterized long non‐coding RNAs that are transcribed from the anti‐sense strand of, and partially overlap with, the alpha‐fetoprotein (AFP) gene. AFP is highly expressed in fetal liver, but its expression decreases to a very low level after birth. In adult liver, AFP expression increases during regeneration due to liver damage or during the onset of hepatocellular carcinoma. Our study has shown that anti‐sense AFP (asAFP) transcripts are alternatively spliced and their abundance is inversely correlated with AFP mRNA during liver development. While these asAFP RNAs had not been annotated, RNA‐seq data from mouse liver confirmed and extended our characterization and ENCODE data for histone modifications and DNase I hypersensitivity show signals near the asAFP exons we have identified. Expression levels of AFP mRNA and asAFP RNA also are inversely correlated in the presence or absence of the Zinc fingers and homeoboxes 2 (Zhx2) gene. Zhx2, a putative transcription factor, was shown previously to contribute to the developmental repression of AFP; in the absence of Zhx2, AFP mRNA is high while asAFP RNA is low, compared to wild‐type. Transiently transfecting asAFP into a liver cell line causes a decrease in endogenous AFP, showing asAFP contributes to AFP regulation in trans. We have shown that the asAFP transcripts localize mainly in the poly(A)+ fraction of cytoplasmic RNA, but this does not identify where the asAFP RNA functions. To determine whether the asAFP RNA represses AFP mRNA in the nucleus or cytoplasm, we have been analyzing the abundance of these two RNAs in nuclear and cytoplasmic RNA from liver of Zhx2 wild‐type or knock‐out animals. To better understand the role of Zhx2 in modulating AFP and asAFP RNA expression, future studies will use chromatin IP to determine whether Zhx2 interacts directly with these genes. To identify which of the genes that are mis‐regulated in the absence of Zhx2 are direct targets, we plan to do Zhx2 ChIP‐seq experiments. Support or Funding Information MCB‐1158234 from the National Science Foundation, R01‐DK59866 from the National Institute of Health