Shape change and Nitric Oxide (NO) Modulation of Core Pluripotent TF Expression

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Our focus shifted during the year to a more unbiased transcriptomic approach. We have performed whole transcriptomic analysis of the genes expressed in 7 day monolayer and spheroid cultures of Clone A and CX-1. The data are still under evaluation but an initial run suggested that there are 462 genes upregulated in both Clone A and CX-1 spheroids with 249 genes up in Clone A but down in CX-1 with 1,195 genes up in CX-1 down in Clone A and 1,861 genes down in CX-1 and Clone A. These changes are out of a total of more than 29,000 genes in the reference sequence list and the addition of several important isoforms of genes that are not present in last refSeq build. We have currently repeated this whole transcriptome analysis as well as to study the effects of inhibiting Nanog expression with specific shRNA to NanogP8 as well as overexpressing NanogP8 in CX-1 cells. These results are still under analysis. In addition, we have begun a collaboration with the Hager lab to identify whether nucleosomes shift location during shape change so that transcription of NanogP8 is increased. We obtained a NanogP8 promoter reporter from the Tang laboratory at MD Anderson and have demonstrated that during transition from monolayer to growth in suspension as 3-D spheroids the level of the promoter activity increases for NanogP8. Earlier we had shown similar activity increases for a Nanog promoter. Since most colorectal carcinoma cells only produce one or other transcript, we postulate that nucleosome movement may account for lack of transcription of one or other Nanog. Hopefully, we will be able during the coming year to clarify 1) the genes regulated by Nanog and NanogP8 during shape change and 2) a mechanism by which transcription is regulated for these two loci.