YY1 Promotes SCF/c-Kit Signaling and HSC Self-Renewal in Fetal Hematopoiesis

Fetal hematopoietic stem cells (HSCs) exhibit markedly different properties as compared to adult HSCs including cell surface marker expression, proliferation state and repopulation capacity. Changes of HSC activity in postnatal mice is defined by a process of decreasing in cell cycle and entering into quiescence. Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group (PcG) Protein with important functions to regulate embryonic development, lineage differentiation and cell proliferation. While homozygote deletion of YY1 in mice results in lethality at the peri-implantation stage, heterozygote deletion of YY1 causes severe developmental defects. By conditionally deleting YY1 in adult hematopoietic system, our previous study showed that YY1 is an essential regulator for adult hematopoiesis by promoting HSC long-term self-renewal and maintaining adult HSC quiescence. In contrast to adult HSCs, in which quiescence is a fundamental characteristic, over 95% of fetal HSCs are in an active cell cycle to rapidly generate homeostatic levels of blood cells for oxygen transport and immune system development in the growing organism. Herein, we assessed whether YY1 was also required for maintaining fetal HSC pool and regulating fetal HSC functions, and what was the underlying mechanism by which YY1 regulated fetal HSCs.

To test how loss-of-function of YY1 impacted fetal hematopoiesis, Yy1^f/f mice in which the Yy1 promoter region and exon 1 are flanked by loxP sites, were crossed to Vav-Cre mice to generate heterozygous Yy1^f/+ Vav-cre mice. The Vav promoter drives Cre recombinase expression specifically in fetal liver hematopoietic cell starting at day E11.5. Yy1^f/+ Vav-cre mice were then subsequently bred with Yy1^f/f mice to generate homozygous Yy1^f/f Vav-cre mice. Among 141 pups resulting from breeding Yy1^f/fto Yy1^f/+ Vav-Cre, only 7 were Yy1^f/f Vav-Cre (n=7) and was significantly lower than the estimated number (n=35) according to the Mendelian ratio (P<0.05). All Yy1^f/f Vav-Cre pups died within 72 hours after birth, which supported essential role of YY1 in fetal hematopoiesis and survival. At E14.5 of fetal development, Yy1^f/f Vav-Cre fetuses had reduced numbers of hematopoietic stem and progenitor cells in the liver. In addition, YY1 deficient fetal HSCs failed to self-renew in primary and secondary bone marrow transplantation assays. Colony formation assay showed that fetal liver cells from Yy1^f/f Vav-Cre mice failed to form CFU-GEMM, CFU-GU and BFU-E compared to Vav-Cre control. While YY1 promotes SCF/c-Kit signaling in adult HSCs, it does not impact c-Kit cell surface expression in early T cell progenitors (unpublished data). To assess YY1 impact on SCF/c-Kit axle in fetal HSCs, c-Kit transcript level, c-Kit median fluorescence intensity and phosphorylated AKT were measured. Similar as its function in adult HSCs, YY1 deficient fetal HSCs had decreased Kit transcript expression, decreased c-Kit cell surface expression and decreased SCF/c-Kit signaling. Our results supported that YY1 is required for maintaining a continuous pool of HSCs in fetal liver and is critical for fetal HSC long-term self-renewal and differentiation. Similar as its function in adult HSCs, YY1 promotes SCF/c-Kit signaling in fetal HSCs.