Analysis of full-length transcripts of the Growth Hormone transcription factor ZNF2929 (Zn16) and circular RNA production

• Main Authors: Josey, Devin, Gregory, Taylor, Bancroft, Alexa, Barnes, Bridget, Hodge, Claire, Nelson, Rachel, Scott, Emily, Watters, Kayla, Zysk, Stacey, Hurley, David L
• Published: Digital Commons @ East Tennessee State University 2019
• Subjects: ZNF2929; Growth Hormone; Pit-1; pituitary; gene expression; Cell Lines; Endocrine System; Cancer or Carcinogenesis
• Online Access: https://dc.etsu.edu/asrf/2019/schedule/133

Growth hormone (GH) is a vital pituitary hormone controlling somatic cell growth and development. GH has a multitude of effects on the body: deficiency can lead to dwarfism while excess can cause conditions such as gigantism. Human patients with mutations in the transcription factor Pit-1 show decreased GH and prolactin levels. However, Pit-1 is known to control multiple pituitary hormones, so what other factors lead to the specificity of transcriptional regulation of the GH gene via its promoter? In order to study this, our lab has been analyzing rat pituitary cell lines to understand the role of ZNF292 (formerly called Zn-16), a selective GH transcription regulator with 16 zinc fingers that bind to the GH promoter DNA. This work has used rat MtT/S cells that are unique in that they exclusively express GH. MtT/S cells were procured from Riken Cell Bank in Japan, cultured, and examined for GH hormone and RNA expression. Results confirmed that the MtT/S cells are terminally differentiated as somatotrophs. To understand the role of ZNF292 in transcription of the GH gene, recent rat genomic data was analyzed to determine the positions of 7 exons upstream from the large exon 8 that contains the important zinc finger-encoding portions of the protein. First, MtT/S RNA was reverse transcribed into DNA, then PCR amplification was performed using primer pairs encompassing various sections of the exon 1 – 7 region. Specific PCR products were found with distinct products ranging in size from 130 to 960, all of which agreed with the predicted sizes of these exons. It had previously been theorized that ZNF292 contained a single large exon; however, these results show that splicing of the primary transcript does occur in this upstream region. Characterizing this exonic region was performed because it has been shown that ZNF292 produces circular RNA (circRNA) of unknown function in human endothelial cells and in certain types of cancer. CircRNAs are thought to be created by the “back-splicing” of exons, so that rather than a linear transcript, the ends are circularized for a portion of the transcript. Having determined the sequence and organization of these upstream exons, we are now testing primer sets that will demonstrate productive amplification only from circRNAs. Further, we are removing linear RNAs using RNAse R treatment to selectively enhance circRNA presence in the reactions. Finally, data from RNA-Seq analysis of the MtT/S cells will be scrutinized to determine if additional exon/intron boundaries or alternative splice sites exist in this upstream 7 exon region. The study of circular RNAs could be very important in understanding its role in acting as a microRNA sponge or RNA-binding protein sponge during their regulation of downstream gene transcription. Also, analysis of this mechanism shows potential as a clinical tool in cancer treatment because ZNF292 functions as a tumor suppressor in colorectal and chronic lymphatic leukemia.