Steward Laboratory

Stem cells, Hematopoiesis, and Epigenetic Control of Chromatin Integrity in Drosophila

Zfrp8, a new gene functioning in stem cells and hematopoiesis in Drosophila

We have identified a new gene, Zfrp8 (PDCD2 in vertebrates) that regulates hematopoiesis in Drosophila. The Drosophila and human proteins are 38% identical. Several loss-of-function alleles of Zfrp8 cause enormous hyperplasia of the hematopoietic organs, the lymph glands, abnormal differentiation of immature blood cells, and severe growth delay in other tissues. The size of the lymph gland is already double that of wild type in late embryos. We have shown that the gene has a function in the regulation of the cell cycle, rather than apoptosis, as proposed by others.

We find that normal lymph gland development is more closely related to hematopoiesis in vertebrates than previously thought and hence represents an excellent model to study differentiation of blood cells. Using clonal analysis we determined that lymph glands contain stem cells at the top of a lineage ultimately giving rise to the three distinct hemocytes. With the same approach we showed that Zfrp8/PDCD2 is essential for the maintenance of the stem cells. We have also established that Zfrp8/PDCD2 function is required in all stem cells investigated so far, namely ovary germline and somatic stem cells, as well as gut stem cells.

The function of PDCD2 in humans is unknown and to gain more insight we are collaborating with Dr. Dale Schaar from the Cancer Institute of New Jersey. We have recently determined that PDCD2 is expressed in hematopoietic stem or precursor cells. In bone marrow and white blood cells from leukemia patients we detect a significant increase of PDCD2 expression compared to normal bone marrow and white blood cells. We have gained approval of an IRB protocol that allows us to study PDCD2 levels in patients. We aim at possibly developing a new disease marker.

In a genetic modifier screen we identified ~12 genes that function in the lymph gland and hemocyte development. We have selected a three of these genes for further analysis.

We have isolated the Dlg5 (Disc large 5) protein as a Zfrp8 interactor in a yeast two-hybrid screen, and also as part of a Zfrp8 protein complex in tissue culture cells. So far we have ascertained that the gene functions in lymph gland development. Further experiments investigating the genetic interaction of dlg5 and Zfrp8 and defining the function of dlg5 in stem cells are under way. Mouse dgl5 controls asymmetric cell division in mouse kidneys and brains.

Epigenetic Control of Chromatin Organization

Chromatin organization is influenced by post-translational modification of the four histones that organize the packaging of DNA into nucleosomes, the basic unit of chromatin

The functionally important modification of lysine 20 of histone H4 is the monomethyl mark set by the PR-Set-7 histone methyl transferase (HMT). Homozygous PR-Set7 mutants die at the larval-to-pupal transition and show reduction in cell number of imaginal discs, indicative of a defect in cell cycle progression.  We have further defined the importance of histone H4-K20 monomethylation by studying the phenotype in a diploid tissue, third-instar larval neuroblasts. Our results establish that in the mutant, several aspects of the cell cycle are abnormal and the DNA damage checkpoint activated. PR-Set7 controls de novo methylation of H4K20 and the methyl mark is stable over several cell generation. We are investigating the role of PR-Set7 in ovary stem cells.

Our work suggests that mono-methylation of histone H4 lysine 20 is essential for higher order chromatin structure that, in turn, is essential for proper chromosome organization. Our results support a basic role of histone methylation in chromatin organization.

Figure: The Drosophila lymph gland is a multi lobed organ (confocal projections above, cross sections below). The primary, largest lobe comprises a medulla, containing undifferentiated cells (blue or dark gray), and a cotex, where the differentiated cells reside (green, plasmatocytes; red and white, crystal cells).