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

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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.