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

该项目的目的是了解转录调节因子FUSE结合蛋白1 （FUBP1）在长期造血干细胞（lt - hsc）自我更新中的基本作用。lt -造血干细胞可引起所有成熟血细胞系的终身再生。FUBP1通过与单链DNA元件FUSE结合并与基础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，并证明它是通过抑制促凋亡和抗增殖靶基因来促进肝细胞癌生长所必需的。此外，我们对两种功能敲除小鼠模型的初步分析发现了FUBP1在LT-HSC自我更新中的一个意想不到的基本功能，这将是本项目进一步研究的内容。工作计划的目标包括确认和分析p21和Noxa作为FUBP1靶基因，其在FUBP1缺陷lt - hsc中的上调解释了这些细胞体外增殖减少和细胞死亡率增加以及它们在体内灭绝的原因。我们的初步分析还显示，在fubp1缺陷的lt - hsc中，三磷酸异构酶1 （TPI1）显著下调。我们将使用细胞培养实验和建立条件Tpi1敲除小鼠模型来验证我们的假设，即Tpi1是lt - hsc厌氧糖酵解和维持干性所必需的。这将增加通过TPI1的代谢调节，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 - hsc中FUBP1下游信号的分析，我们将通过对亲本和FUBP1缺陷lt - hsc的RNA测序进行转录组范围的分析。这一系统分析将使我们能够鉴定出与LT-HSC自我更新相关的新的FUBP1靶基因。我们的研究结果将有助于我们了解FUBP1作为单链DNA的传感器和转录网络的调节器，同时影响几个细胞命运决定，以确保和使LT-HSC自我更新。

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.