Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin − , c-kit HI , Sca-1 HI , IL-7Rα − (KSIL) cells and Ter119 + erythroid cells. To define Hmgb3 function, we generated hemizygous mice ( Hmgb3 −/Y ) using 129/SvJ ES cells. Hmgb3 −/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3 −/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin − , Sca-1 + , c-kit − cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3 . Prior to 5-FU treatment, 27% of KSIL cells were GFP + (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin − , c-kit − , Sca-1 + cells were GFP + before 5-FU treatment whereas 26% of lin − , c-kit − , Sca-1 + cells were GFP + 4 days p.i. The increased proportion of GFP + lin-, c-kit − , Sca-1 + cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit −/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3 −/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3 −/Y mice contained similar numbers of bone marrow cells (7 x 10 6 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 10 6 cells/hind limb and KSIL cells were still absent. However, in Hmgb3 −/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 10 6 cells/hind limb, an increase of 43% compared to WT (p Hmgb3 −/Y lin − , c-kit − , Sca-1 + population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin − , c-kit − , Sca-1 + cells sorted from 4 day p.i. 5-FU treated Hmgb3 −/Y mice. Recipients received either 2 x 10 4 KSIL or 2 x 10 5 lin − , c-kit − , Sca-1 + cells (Ly 5.2) from 5-FU treated Hmgb3 −/Y mice along with a radioprotective dose of 3 x 10 5 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited Hmgb3 −/Y KSIL cells. There was no detectable engraftment of Hmgb3 −Y lin − , c-kit − , Sca-1 + cells. In contrast to WT mice, both KSIL and lin − , c-kit − , Sca-1 + cells from 5-FU treated Hmgb3 −/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kit HI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.