Hematopoietic Stem Cell Function Is Regulated By Hormonal and Epigenetic Factors

Gender-specific hormones have been known to play a role in hematopoietic function for some time. For example, treatment with estrogens suppresses B lymphocyte production in murine bone marrow, and hormonally compromised mice undergoing hematopoietic stem cell transplantation demonstrate enhanced immune reconstitution. Furthermore, androgens have been employed as therapy for bone marrow failure syndromes. Despite these experimental observations and clinical practices, the precise molecular mechanism by which gender-specific hormones influence physiology is not understood.

To test if epigenetic modifications could alter HSC function in a gender-specific manner, we compared the engraftment potential of hematopoietic stem cells (HSCs) with altered DNA methylation patterns in female versus male recipients. We used DNMT3B7 transgenic mice as the HSC source. Our laboratory demonstrated that the introduction of DNMT3B7, a truncated DNMT3B isoform commonly expressed in cancer cells, impedes normal embryonic development. Homozygous DNMT3B7 transgenic mice have developmental defects similar to the Immunodeficiency, Centromeric instability, Facial anomalies syndrome, and demonstrate lymphopenia and defective craniofacial development. These physiological defects are accompanied by global DNA hypermethylation and disruption in DNA methylation patterns (Shah MY et al, Cancer Res. 2010). Since DNMT3B7 homozygous mice fail to survive past the day of birth, we used a transplantation model to assay the effect of DNMT3B7 on hematopoiesis.

We found large differences in engraftment potential when cells expressing DNMT3B7 were transplanted into female versus male recipients. Pancytopenia occurred at two weeks, with anemia and leucopenia persisting until eight weeks post-transplantation when females received DNMT3B7 homozygous cells. However, cells from wild-type (WT) embryos engrafted normally regardless of recipient gender. We also observed that oophorectomized female recipients engrafted DNMT3B7-expressing cells normally. Interestingly, we found an improved engraftment of WT cells in these oophorectomized mice, suggesting that female hormones repress hematopoiesis. In competitive transplantation experiments to determine HSC function, the CD45.1 and CD45.2 alleles were used to distinguish competitor and experimental cells respectively. We observed that DNMT3B7-expressing CD45.2⁺ cells were out-competed by WT CD45.1⁺ cells within female recipients, although there were 4-fold more transgenic cells than CD45.1⁺ competitor cells. Because our previous studies suggested that DNMT3B7 functions as a dominant negative isoform of Dnmt3b, we compared our results with DNMT3B7-expressing cells to those observed with competitive transplants using Dnmt3b knockout cells. Cells from WT, heterozygous Dnmt3b, and homozygous Dnmt3b knockout embryos had similar engraftment potentials in female recipients and were not out-competed by competitor WT CD45.1+ cells, similar to previous observations in a distinct Dnmt3b knockout mouse model (Challen GA et al, Nat Genet. 2011). DNMT3B7 homozygous embryos had significantly fewer numbers of HSCs than WT embryos, as assayed by the LSK (Lineage-, Sca1+, Kit+) and SLAM (CD48, CD150) set of markers. We observed a dose-response relative to DNMT3B7 content, with DNMT3B7 homozygous embryos having the fewest number of HSCs, and DNMT3B7 hemizygous embryos having intermediate numbers of HSCs compared to WT embryos.

These observations point to the dual influence of epigenetics and hormones on HSC function. Our hope is that we will be able to use our understanding of the molecular basis for the influence of hormonal milieu on hematopoiesis to augment stem/progenitor cell function in patients undergoing stem cell transplantation and chemotherapy.