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

核心多能 TF 表达的形状变化和一氧化氮 (NO) 调节

• 批准号: 8349390
• 负责人: John Jessup
• 金额: $ 6.08万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349390
• 关键词: 3-Dimensional; Accounting; Acetylation; Adult; Affect; Architecture; Area; Cell Shape; Cells; Chromatin; Collaborations; Cyclic GMP; DNA Sequence Rearrangement; Data; Evaluation; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Growth; Guanylate Cyclase; Histones; Laboratories; Large Intestine Carcinoma; Location; Maintenance; Malignant Epithelial Cell; Maps; Methylation; Movement; Nitric Oxide; Nucleosomes; Pathway interactions; Pattern; Physiological; Protein Isoforms; Proteins; Reporter; Research Personnel; Role; Running; Shapes; Signal Transduction; Signaling Molecule; Suspension substance; Suspensions; Transcript; chromatin remodeling; induced pluripotent stem cell; monolayer; nerve stem cell; nitration; overexpression; promoter; small hairpin RNA; therapy resistant; transcriptomics; tumor progression

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.

我们的重点在一年中转变为一种更加偏见的转录组方法。我们已经对克隆A和CX-1的7天单层和球体培养物表达的基因进行了整个转录组分析。数据仍在评估中，但初步运行表明，克隆A和CX-1球体中有462个基因上调，克隆A中有249个基因，但在CX-1中向下，但在CX-1中向下降低，CX-1中的1,195个基因在CX-1中向上下调了CX-1中的1,195个基因，在CX-1中向下降低了CX-1和1,861个基因的CX-1和1,861个基因。这些变化是CX-1和Clone A的其他重要性。最后一个refseq构建中不存在的基因的同工型。 我们目前已经重复了整个转录组分析，并研究了抑制特异性shRNA对NanogP8的纳米表达以及CX-1细胞中的NanogP8过表达的影响。这些结果仍在分析中。 此外，我们已经开始与Hager Lab进行合作，以确定核小体在形状变化过程中是否移动位置，从而增加NanogP8的转录。我们从MD Anderson的Tang实验室获得了NanogP8启动子报告基因，并证明在从单层到悬浮液中的过渡期间，随着3-D球体的过渡，NanoGP8的启动子活性水平增加。早些时候，我们显示了纳米启动子的类似活性增加。由于大多数结直肠癌细胞仅产生一个或另一个转录本，因此我们假设核小体运动可能是一个或其他Nanog的转录。希望我们能够在来年澄清1）在形状变化期间由Nanog和Nanogp8调节的基因，以及2）对这两个基因座进行转录调节的机制。