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Shigeru Sakonju

Associate Professor of Human Genetics

B.A. Columbia Union College

Ph.D. Johns Hopkins University

Research

References

ssakonju@genetics.utah.edu

 

Research

We study the regulation of homeotic gene expression in the fruit fly Drosophila melanogaster . Homeotic genes are master regulatory genes, conserved from flies to humans, that specify identities of body segments. Each homeotic gene is expressed in a unique body domain along the body axis and directs cells to follow an appropriate developmental pathway for their position. If a homeotic gene is misexpressed outside of its normal domain, it transforms the misexpressing body segments to look like another segment. Some spectacular examples are antenna transformed into legs, or four-winged flies. These homeotic transformations are not just developmental curiosities, but serve as easily observable and sensitive phenotypes that we can use to study how homeotic genes are regulated. By understanding the regulation of homeotic genes, we will be learning how bodies of higher organisms, including that of humans, are specified during development. It also provides insight into the molecular basis of other developmental defects and diseases caused by misregulation of gene expression.

The current focus of our lab is to understand how silencing of homeotic genes is maintained. The maintenance of silencing relies on a group of chromatin-associated proteins, called Polycomb group, or PcG, proteins. These proteins are present in all cells throughout the body, but they recognize and silence only those homeotic gene promoters that had been repressed during early embryogenesis by transiently present proteins called gap proteins. Silencing of these promoters is then faithfully transmitted through cell divisions during development, while the promoters not repressed by gap proteins remain active despite the presence of PcG proteins. The maintenance of silencing by PcG proteins is therefore epigenetic, meaning that features that distinguish silent from active promoters are not encoded in DNA sequences but are in something else, such as chromatin structure. To establish silencing by PcG proteins, however, cis-regulatory elements called Polycomb Response Elements, or PREs, are required. We have been interested for some time in understanding what proteins bind to these elements, and how and when PcG proteins are recruited to their targets through PREs. We have shown that two proteins, Pleiohomeotic and GAGA factor, bind to the PRE that we have analyzed. These proteins appear to recruit large PcG protein complexes through protein-protein interactions. It is not yet clear exactly what the recruited PcG proteins do to silence promoters. One biochemical activity of PcG complexes that has been identified by other labs is methylation of histone H3, one of the four constituent proteins of nucleosomes. Thus modification of a histone appears to contribute to a chromatin structure that prevents gene expression. During the last few years, we have been doing experiments to examine if recruitment of PcG proteins through a PRE must occur after each cell division to keep a promoter silent, or chromatin modifications by PcG proteins are sufficient to epigenetically maintain silencing. To answer this question, we have devised a novel clonal analysis method and found that PcG proteins do not need to be recruited through PREs after each cell division. Rather, most critical recruitment of PcG proteins through PRE binding proteins occurs during embryogenesis when they are first recruited to the promoters to be silenced. Thereafter PcG proteins are able to continue their association with silent promoters through other means.

We are also studying novel pathways that influence silencing of homeotic genes. For example, we have examined the effect of genes that are known to contribute to another form of silencing called position effect variegation or PEV. PEV silencing is observed when a gene normally located in euchromatin (gene-rich regions of chromosomes) becomes juxtaposed to heterochromatin (gene-poor regions near centromeres and telomeres). Heterochromatin comprises repetitive and other "junk" DNA sequences, and is rich in silencing proteins which keep these sequences from being expressed.   It has been thought that PEV and PcG-mediated silencing are two separate processes. We have been accumulating evidence that they are not completely different but appear to use at least some common mechanisms. For example, a distinguishing feature of PEV has been its sensitivity to the amount of heterochromatin in the genome; the level of silencing can be strengthened or weakened by decreasing or increasing the amount of heterochromatin. We found that this sensitivity to heterochromatin is also observed in homeotic gene silencing by PcG proteins. We also discovered that some genes that affect PEV silencing also contributes to homeotic gene silencing as well. We are particularly interested in one such gene, called Su(var)2-10, because this gene is implicated in a pathway that modifies proteins by a small ubiquitin-like peptide called SUMO. One of the biochemical effects of protein modification by SUMO has been its role in assembly of proteins in certain nuclear compartments. There are several genetic and cytological observations that suggest that nuclear compartments may play a role in the regulation of homeotic gene expression. By genetic interaction studies, we have shown that the SUMO pathway in Drosophila does indeed affect homeotic gene silencing. We have also found by in vitro studies that at least one PcG protein can be modified by SUMO and that it forms complexes with enzymes in the SUMO modification pathway. By elucidating the effect of this modification pathway on homeotic gene expression, we are aiming to uncover a level of gene regulation that involved three-dimensional aspects of the nucleus and goes beyond transcription factor-promoter interactions.

References