Runx1 Induced Chromatin Folding of the PU.1 Gene Locus Is Necessary for Adult Long-Term Hematopoietic Stem Cell Maintenance

PU.1 is a hematopoietic transcription factor that is essential for normal hematopoiesis. PU.1 gene expression is mainly regulated through the proximal promoter (PrPr), and an upstream regulatory element (URE) located –14kb or –17kb upstream of the transcription start site in mice and humans, respectively. We previously demonstrated that PU.1 is a downstream target of core binding factor Runx1 (also called AML1) for megakaryocytic, lymphoid, and myeloid differentiation. Recent reports identified Runx1 to restrict proliferation of hematopoietic stem cells (HSCs) and to prevent HSC exhaustion.

By employing conditional (Mx1-Cre) Runx1 knockout mice (Runx1^Δ/Δ) and knock-in mice with targeted disruption of all three Runx1 binding sites at the −14kb URE of PU.1 (mRunx^ki/ki) we aimed to genetically dissect a potential functional role and mechanisms by which Runx1 might regulate PU.1 expression in HSCs.

Both, Runx1^Δ/Δ and mRunx^ki/ki mice exhibited decreased PU.1 levels of approximately 65% in purified phenotypic HSCs (CD150+CD48-LSKs). In addition, activating histone marks H3K4me3 and H3Ac were significantly decreased at the −14kb enhancer and the PrPr. Mechanistically, we found by quantitative chromosome conformation capturing (3C) that Runx1 binding established a physical interaction of the −14kb enhancer with the PrPr thereby forming an active chromosomal conformation for proper PU.1 transcription. In concordance to Runx1^Δ/Δ, phenotypic HSCs of mRunx^ki/ki mice were significantly increased in number. However, limiting dilution transplantations using the congenic Ly5.1/Ly5.2 system revealed a 25-fold decrease of competitive repopulating units (CRU) in phenotypic HSCs of mRunx-ki/ki mice compared to controls. Long-term reconstitution in serial transplantation assays was also severely impaired indicating HSCs exhaustion in mRunx^ki/ki mice. HSC homing and engraftment was not affected. Importantly, restoration of PU.1 levels by crossing to a human PU.1 transgenic fully rescued long-term HSC functions, thus demonstrating that HSC maintenance was strictly related to PU.1 levels.

Runx1 binding to the −14kb enhancer of PU.1 is necessary to maintain an active chromosomal conformation for proper PU.1 transcription in order to prevent HSC exhaustion.

No relevant conflicts of interest to declare.