The Rel Pathway, dorsal-ventral Patterning, and hematopoiesis in Drosophila

The Toll-Dorsal (NF-kB/Rel) pathway functions in establishing dorsal-ventral polarity in the early Drosophilaembryo and in the humoral and cellular immune response. The pathway is conserved in flies and vertebrates. It functions in mammals in the immune and inflammatory responses and is critical for cell growth and survival. A large number of mammalian tumors are associated with mis-regulation of the NF-kB/Rel proteins.

Dorsal, like all other Rel proteins, is retained in an inactive state in the cytoplasm through direct interaction with the IkB protein Cactus. The ventral signal, transmitted through the transmembrane receptor Toll, controls the formation of a ventral-to-dorsal nuclear Dorsal gradient, resulting in the formation of the dorsal-ventral axis.

Mis-regulation of the NF-kB/Rel pathway also results in overgrowth of hematopoetic cells and melanotic nodules in Droophilia.  We are studying two genes involved in the formation of melanotic tumors.  One, cactin, was initially identified through itsinteraction with the IkB protein, Cactus.  We produced transgenic flies expressing a cactin-RNAi construct in hematopoetic cells and fat body and we find that their larvae produce melanotic tumors similar to cactus loss-of-function larvae.  We have isolated a null allele of cactin by gene targeting and find that it is an essential gene; homozygous larvae are lethal in early second instar.  The gene is also essential in germline cells during oogenesis.  We are further investigating the cellular function of cactin. 

The other melanotic nodule gene is zfrp8 (PDCD2 in vertebrates). The Droophilia and human proteins are 38% identical.  We found that in Droophilia the PDCD2/zfrp8 gene functions, most probably as an intrinsic factor, in blood cell proliferation. 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.

The function of PDCD2 in humans is unknown and to gain elucidation we have initiated a collaboration with Dr. Dale Schaar from the Cancer Institute of New Jersey. On western blots we find that the protein is absent in non-proliferating tissues like the hypocampus and mature blood mono-nuclear cells.  In normal bone marrow, we detect a 48kD protein.  In white blood cells from leukemia patients and in all human tissue culture cell lines tested so far, we detect a 44kD PDCD2 form. Importantly, in bone marrows from leukemia patients, we detect both the 48kD and the 44kD bands.  The intensity of the 44kD band appears to correlate with the disease state of the patient. The PDCD2/Zfrp8 proteins contain a zinc-finger domain, often found in DNA binding proteins, and a PDCD domain found in only two proteins in vertebrates and flies. We have shown in flies that the gene is essential for the control of cell proliferation.

Epigenetic Control of Gene Expression

The development of a single cell into an embryo consisting of specific tissues is dependent on cascades of cell-to-cell signaling events that activate transcription factors controlling the expression of specific genes. Control of expression of these genes is regulated at two levels, the interplay of transcription factors that bind DNA directly, and the conformation of chromatin that controls the access of the transcription factors to the DNA.  Chromatin organization is controlled, at least in part, by post-translational modification of the four histone proteins that organize the packaging of DNA into the nucleosome, the basic unit of chromatin.

While the trimethyl mark appears to be the most important modification of lysines in other histones, the functionally important modification of lysine 20 of histone H4 is the monomethyl mark.  Droophilia has one Suv4-20 gene.  We have produced a null allele of Suv4-20 and have shown that in this mutant di- and trimethylation of H4-K20 is not detected, but that the level of monomethylated H4-K20 is significantly increased.  This suggests that monomethylated H4-K20 is the substrate for Suv4-20 and that this enzyme can both di- and trimethylate H4-K20. 

We have looked for a phenotype associated with the loss of Suv4-20 function and find that the gene is redundant for viability of our flies, and that development fertility, meiosis, and the DNA damage response are normal.  Nor could we document a function of Suv4-20 in the suppression of position effect variegation (PEV), as previously reported by others.

We are studying the distribution and function of the PR-Set-7 histone methyl transferase (HMT) in Drosophila development and have produced a complete loss of function mutation in the Pr-Set7 gene. In homozygous mutants, the maternally deposited PR-Set7 protein does not perdure into the first instar larval stage, but mono-, di-, and trimethylation of H4-K20 is detectable into late larval stages when all three methyl marks are reduced.

PR-Set7 mutants suppress PEV, confirming that PR-Set7 functions in silencing gene expression. Themutants die at the larval-to-pupal transition and show strong phenotypes in their imaginal discs; the number of cells in the discs is reduced.

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 show that in the mutant, several aspects of the cell cycle are abnormal and that, surprisingly, the DNA damage checkpoint is activated.  Some of the mitotic defects are restored in a double mutant in PR-Set7 and the DNA damage checkpoint protein mei-41 (the fly ATR ortholog).

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

These insights are novel because most histone methylattions, particularly the methylation of histone H4-K20, have generally been considered to be regulators of transcription.

Recent Publications

Sakaguchi, A., and Steward, R. Aberrant mono-methylation of histone H4 lysine 20 activates the DNA damage checkpoint in Drosophila.J. Cell Biol. In press.

Karachentsev, D., Minakhina, M., and Steward, R. Free and chromatin-associated mono-, di-, and trimethylation of histone H4-lysine 20 during development and cell cycle progression. Devel. Biol. In press.

Minakhina S, and Steward. Melanotic mutants in Droophilia: pathways and phenotypes. Genetics 174: 253-63 (2006)

Minakhina S, and Steward R. Nuclear factor-kappa B pathways in Droophilia. Oncogene 30:6749-57 (2006)

Minakhina, S., Meyers, R., Druzhinina, M., and Steward, R.  Crosstalk between the actin cytoskeleton and Ran-mediated nuclear transport. BMC Cell Biol. 24:6:32 (2005).

Silber, K., Serr, M., Steward,  R., Hays T.S., and Doe, D.Q.  A Lis1/dynactin protein complex independently regulates spindle positioning and mitotic checkpoint inactivation. Mol Biol Cell. 16:5127-40 (2005).

Minakhina S, and Steward R.  Axes formation and RNA localization. Curr Opin Genet Dev. 4:416-21 (2005)

Bin Yu, Zhiyong Yang, Junjie Li, Svetlana Minakhina, Maocheng Yang, Richard W. Padgett, Ruth Steward, and Xuemei Chen. Methylation as a crucial step in plant microRNA biogenesis.  Science 307:932-935. (2005).

Karanchentsev, D. , Sarma, K.,  Reinberg,  D., and Steward, R. PR-Set7 dependent Methylation of Histone H4 Lysine 20 is a cell cycle specific mark and is essential for mitosis. Genes & Development 19:431-435. (2005).

Reinberg, D., Vhuikov, S., Farnham, P., Karachentsev, D., Kirmizis, A.,  Kuzumichev, A., Marguerson, R., Nishioka, K., Preissner, T.S., Sarma, K., Abate-Shen, C., Steward, R., and Vaquero, A. Steps towards understanding the Inheritance of Repressive Methyl-Lysine marks in Histones. Cold Spring Harbor Symposium on Quantitative Biology 69: In press. (2004)