Expression of a Truncated DNMT3B Protein, DNMT3B7, Highlights the Sensitivity of Hematopoietic Progenitor Cell Function to Hormonal Milieu.

Abstract 83

Epigenetic changes, including DNA methylation and histone modifications, alter chromatin structure and regulate gene transcription in numerous cellular processes, including stem cell differentiation, mammalian embryogenesis, genomic imprinting, X-chromosome inactivation, and in cancer cells. Our laboratory studies the molecular basis for the abnormal distribution of DNA methylation in tumors. We found that cancer cells exhibit aberrant splicing of the DNMT3B gene, which encodes one of the de novo DNA methyltransferase enzymes. Aberrant DNMT3B transcripts encode truncated proteins, some of which lack the C-terminal catalytic domain. We hypothesize that aberrant DNA methylation in cancer cells is due in part to the presence of these truncated, catalytically inactive DNMT3B proteins.

To test the in vivo effects of expression of a truncated DNMT3B protein, we engineered transgenic mice to express DNMT3B7, a truncated isoform expressed in cancer cells, and tested its influence on murine hematopoiesis. Since homozygous DNMT3B7 transgenic mice die in mid-gestation or within hours of birth, we propagated transgenic fetal liver cells (FLCs) in lethally irradiated recipients to bypass the animals' developmental abnormalities. DNMT3B7 was expressed in E14.5 FLCs, and we achieved approximately 80% donor chimerism in all recipients. Cells from wild-type (WT) embryos engrafted normally regardless of recipient gender. However, pancytopenia occurred at 2 weeks, with anemia and leucopenia persisting until 8 weeks post-transplant when females received DNMT3B7 homozygous cells. Male recipients displayed normal peripheral blood counts regardless of donor cell genotype. For example, females receiving WT or hemizygous cells had hemoglobin levels of 10 g/dL, whereas those receiving homozygous cells had levels of about 6 g/dL. Anemia was not seen in male recipients, where hemoglobin levels were 11-12 g/dL across all donor genotypes. When DNMT3B7 homozygous cells were transplanted into female recipients, neutropenia and lymphopenia were observed. Normal white blood cell recovery was seen in male recipients. Additionally, thrombocytopenia was observed at 2 weeks in female recipients of homozygous DNMT3B7 transgenic cells, but the platelet count normalized in these animals by 4 weeks.

In preliminary experiments to further examine the role of hormonal milieu, oophorectomized female recipients demonstrated loss of the previously observed effect. At 2 weeks, oophorectomized females transplanted with DNMT3B7 homozygous cells showed recovery of hemoglobin levels to levels around 11 g/dL, the same level seen in normal female recipients transplanted with WT or hemizygous cells. Oophorectomized females receiving homozygous cells also showed improvement in white blood cell count. This suggests that the female hormonal milieu is suppressive to DNMT3B7-expressing hematopoietic progenitor cell function, rather than male-specific hormones augmenting hematopoiesis.

We hypothesize that DNMT3B7 alters the DNA methylation patterns, and consequently, the gene expression profiles of hematopoietic progenitor cells, revealing a dependence of these cells on a particular hormonal milieu in recipient animals. Preliminary gene expression profiling of DNMT3B7-expressing versus WT E14.5 fetal liver cells reveals 29 genes are differentially expressed. These genes fall into interesting gene ontogenies, including chromatin modification genes (GSG2, SUZ12), cell cycle, programmed cell death, cell differentiation and proliferation. The defective hematopoiesis seen up to 8 weeks after transplantation in female recipients of DNMT3B7-expressing progenitor cells, suggests that there is an important relationship between progenitor cell function and hormonal milieu. 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.