Cul4A Is Required for Cell Viability and Its Deficiency in Hematopoietic Cells Causes Apoptosis and Is Fatal.

Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A-deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele (Cul4A^flox) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre, were derived. When deletion of Cul4A was induced in Cul4A^flox/flox Mx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls (Cul4A^flox/flox mice without Mx1-Cre), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A-deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A-deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A-deficient hematopoietic cells, deletion was induced in vivo in Cul4A^flox/flox Mx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A-deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A-deficiency will be presented.