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

我们的重点在这一年转移到一个更公正的转录组学方法。我们对克隆A和CX-1的7天单层和球状体培养物中表达的基因进行了全转录组学分析。数据仍在评估中，但初步运行表明，在克隆A和CX-1球状体中有462个基因上调，其中249个基因在克隆A中上调，但在CX-1中下调，其中1，195个基因在CX-1中上调，在克隆A中下调，1，861个基因在CX-1和克隆A中下调。这些变化来自参考序列列表中总共超过29，000个基因，并且添加了上一次refSeq构建中不存在的几种重要基因亚型。 我们目前已经重复了这种全转录组分析，并研究了用NanogP 8的特异性shRNA抑制Nanog表达以及在CX-1细胞中过表达NanogP 8的效果。这些结果仍在分析中。 此外，我们已经开始与Hager实验室合作，以确定核小体是否在形状变化期间移动位置，从而增加NanogP 8的转录。我们从MD安德森的Tang实验室获得了NanogP 8启动子报告基因，并证明了在从单层到悬浮生长为3-D球状体的过渡期间，NanogP 8的启动子活性水平增加。早些时候，我们已经显示了Nanog启动子的类似活性增加。由于大多数结直肠癌细胞只产生一种或其他转录本，我们假设，核小体运动可能是缺乏一个或其他Nanog的转录。希望在未来的一年里，我们能够澄清1）Nanog和NanogP 8在形状变化过程中调控的基因，以及2）这两个基因座调控转录的机制。

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.