Functional analysis of the single-stranded DNA-binding protein FUBP1 as a transcriptional regulator of hematopoietic stem cell self-renewal

单链DNA结合蛋白FUBP1作为造血干细胞自我更新转录调节因子的功能分析

• 批准号: 276833671
• 负责人: Professor Dr. Martin Zörnig
• 金额: --
• 依托单位: Else Kröner-Fresenius-Stiftung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/276833671?language=en
• 关键词: Functional; analysis; single; stranded; DNA

The aim of this project is to understand the fundamental role of the transcriptional regulator FUSE binding protein 1 (FUBP1) in the self-renewal of long-term hematopoietic stem cells (LT-HSCs). LT-HSCs give rise to the life-long regeneration of all mature blood cell lineages. FUBP1 functions as a transcriptional regulator by binding to the single-stranded DNA element FUSE and interacting with the basal transcriptional machinery. We found FUBP1 in a screen for novel anti-apoptotic proteins and demonstrated that it is required for the growth of hepatocellular carcinoma by inhibiting pro-apoptotic and anti-proliferative target genes. In addition, our preliminary analyses of two functional knockout mouse models uncovered an unexpected essential function of FUBP1 in LT-HSC self-renewal that will be further pursuit in this project. The objectives of the work program include the confirmation and analysis of p21 and Noxa as FUBP1 target genes whose upregulation in FUBP1-deficient LT-HSCs explains the decreased proliferation and increased cell death rates of these cells ex vivo and their extinction in vivo. Our preliminary analysis also revealed a significant downregulation of triosephosphate isomerase 1 (TPI1) in FUBP1-deficient LT-HSCs. We will use cell culture experiments and an established conditional Tpi1 knockout mouse model to test our hypothesis that TPI1 is required for anaerobic glycolysis and maintenance of stemness in LT-HSCs. This would add the regulation of metabolism via TPI1 as a further level at which FUBP1 promotes LT-HSC self-renewal. To complement our analysis of FUBP1 downstream signaling in LT-HSCs, we will perform a transcriptome-wide analysis by RNA sequencing of parental and FUBP1-deficient LT-HSCs. This systematic analysis will allow us to identify novel FUBP1 target genes that are relevant for LT-HSC self-renewal. Our results will contribute to our picture of FUBP1 as a sensor of single-stranded DNA and as a regulator of a transcriptional network that simultaneously influences several cell fate decisions to ensure and enable LT-HSC self-renewal.

本项目的目的是了解转录调节因子融合结合蛋白1(FUBP1)在长期造血干细胞(LT-HSCs)自我更新中的基础作用。LT-HSCs可使所有成熟血细胞系终生再生。FUBP1通过与单链DNA元件融合并与基本转录机制相互作用而发挥转录调节作用。我们在筛选新的抗凋亡蛋白中发现了FUBP1，并证明了它是肝细胞癌生长所必需的，它通过抑制促凋亡和抗增殖的靶基因。此外，我们对两个功能性基因敲除小鼠模型的初步分析揭示了FUBP1在LT-HSC自我更新中意想不到的重要功能，这将是本项目进一步研究的方向。该工作计划的目标包括确认和分析p21和Noxa作为FUBP1靶基因，它们在FUBP1缺陷的LT-HSC中的上调解释了这些细胞体外增殖减少、细胞死亡率增加和体内消亡的原因。我们的初步分析还显示，在FUBP1缺陷的LT-HSCs中，磷酸丙糖异构酶1(TPI1)的表达显著下调。我们将使用细胞培养实验和已建立的条件性Tpi1基因敲除小鼠模型来验证我们的假设，即在LT-HSCs中，TPI1是厌氧糖酵解和维持茎的必需的。这将增加通过TPI1对代谢的调节，作为FUBP1促进LT-HSC自我更新的进一步水平。为了补充我们对LT-HSCs中FUBP1下游信号的分析，我们将通过对亲本和FUBP1缺陷的LT-HSCs进行RNA测序来进行转录全组分析。这一系统的分析将使我们能够识别与LT-HSC自我更新相关的新的FUBP1靶基因。我们的结果将有助于我们将FUBP1描述为单链DNA的传感器和转录网络的调节器，该网络同时影响几个细胞命运的决定，以确保和使LT-HSC自我更新。