Harmit Singh Malik - US grants

DESCRIPTION (provided by applicant): Defects in chromosome segregation can lead to infertility, to birth defects and to cancer. Centromeres serve as attachment points of mitotic and meiotic spindles to DMA, and mediate the faithful segregation of all eukaryotic chromosomes. Centromeric DMA evolves rapidly, and can range in size and complexity over several orders of magnitude. Traditional attempts at studying centromeres have left unexplained the causes underlying this complexity and rapid evolution. Our approach is to study the proteins that epigenetically determine centromere identity, instead of directly studying centromeric DNA sequence. We have discovered that the Drosophila centromeric histone (CenH3), Cid, has constantly evolved under positive selection, suggesting its involvement in recurrent genetic conflict. Our hypothesis is that 'centromere-drive' is the source of this conflict. Under this model, centromeres compete via microtubule attachments for preferential transmission in female meiosis in animals and plants, since only 1 of 4 meiotic products becomes the egg. This competition confers a selfish advantage to chromosomes that can make more microtubule attachments, and can result in runaway expansions of centromeric satellites. While beneficial to the 'driving' chromosome, these expansions can have deleterious effects on the fitness of an organism and of the species. For instance, in human populations, Robertsonian fusions (chromosome fusions at centromeres found in 0.12% of the population) are preferentially transmitted through females but male carriers of Robertsonian fusions can be partially or completely sterile. We propose that CenH3s as well as other heterochromatin proteins may be under positive selection to suppress the deleterious consequences of 'centromere-drive' and to restore meiotic parity. We plan to test our hypothesis with the following specific aims: (1) We will test whether recent satellite expansions in D. melanogaster have a transmission advantage in female meiosis, (2) We will examine the effects of the positive selection of Cid and other heterochromatin proteins in Drosophila by replacing 'adapted' endogenous genes with 'unadapted' versions from closely related species or hypothetical ancestors, and (3) We will assay the effects of asymmetric female meiosis on centromere evolution, in yeast and Tetrahymena that lack female and male meiosis, and bdelloid rotifers that lack meiosis altogether.

Mobile genetic elements have resided within eukaryotic genomes for hundreds of millions of years. These mobile elements include retroviruses that integrate into the genomes of their hosts as an essential step in their life cycle. While most such integration events are either deleterious or inconsequential to the host, on rare occasions they can be helpful, the retroviral genes responsible for the useful function can persist, and the function can become established as part of the host's repertoire. This phenomenon is referred to as "domestication" of the retroviral gene. Especially intriguing are those instances of domestication in which retroviral envelope genes remain in the host genome. These envelope genes are the same ones that enable retroviruses to infect the host cells. The genomes of primates and rodents contain domesticated retroviral envelope genes (called "syncytin" genes) that have important roles in placental function. Dr. Harmit Malik, the principal investigator of this project, has shown that a similar domestication event has taken place within the fruit fly Drosophila melanogaster. The domesticated gene, called Iris, was acquired from an insect retrovirus and has been maintained as a host gene for more than 25 million years. Unexpectedly, Iris continues to evolve rapidly whereas previous studies have shown that mammalian syncytin genes do not. The investigator will test two alternate hypotheses to account for Iris's preservation as a host gene. The first hypothesis, that Iris performs a beneficial role in D. melanogaster independent of any retrovirus, i.e., a "housekeeping role", will be tested using a combination of population genetics (fitness experiments), and cell biology (trafficking) experiments. Second, Dr. Malik will test whether Iris may have been domesticated because it protects the D. melanogaster genome against invasions by the insect retroviruses that it originated from, again using a combination of the two methods listed above, as well as fly infection (virology) methods. Finally, using reconstructed evolutionarily ancestral sequences, Dr. Malik will investigate what selective pressures drove the domestication of the Iris gene, starting from its evolutionary origins as an infectious agent. This research plan is the first to explicitly test the evolutionary significance of domesticated genes from retroviruses in a genetically tractable model organism, and is a striking example of "evolution in action." The strength of the proposal stems from an approach that iteratively combines functional and computational methods. In his research Dr. Malik has constantly striven to bridge these two fields to gain further insight into biological processes.

This multidisciplinary approach presents numerous training opportunities at various levels of experience, ranging from those appropriate for starting level researchers (population cage fitness experiments- appropriate for high school students or undergraduates), through those suitable for mid-level researchers (cell biology experiments- suitable for senior undergraduates and graduate students), to those requiring upper-level senior researchers (biochemistry experiments- suitable for graduate students and postdoctoral fellows). To facilitate further functional studies regarding the domestication of retroviral genes, Dr. Malik will establish a comprehensive, public database of recently domesticated eukaryotic genes with ancestries going back to retroelements. There is a pressing need for this kind of resource as well as for this kind of early formal introduction to "functional" evolutionary biology. As part of this project, Dr. Malik and his lab will use the existing infrastructure already in place at the Fred Hutchinson Cancer Research Center (FHCRC) to provide tutorial-based classes in bioinformatic searches primarily for high school science teachers. "Tests" for students, evaluation forms, and direct feedback from the high school teachers will be used to determine the efficacy of material. Tutorials that have been refined by two years of feedback will then be made publicly available to high school teachers across the country. For senior undergraduates and graduate students the investigator will also design and teach a course that explicitly addresses the role of evolutionary antagonism, or genetic conflict, in shaping genomes. Précis versions of this class will be also made available as seminars on the web. Dr. Malik will solicit opportunities to make presentations at primarily undergraduate institutions in the Puget Sound area (Seattle). Members of his lab will also either speak or present posters at conferences for under-represented minorities and women, where the MCB graduate program (jointly run by the FHCRC and Univ. of Washington) actively recruits incoming students. Members of the Malik Lab will also continue to make presentations in the Seattle community to impress upon a general public the role that evolutionary biology plays in the current understanding and practice of medicine.

DESCRIPTION (provided by applicant): Many interferon-stimulated genes (ISGs) and other antiviral mechanisms protect humans from a large variety of viruses. Taken from a viral perspective, many ISGs represent potent barriers to viral replication. Therefore, to be evolutionarily successful, viruses must find ways to evade these host antiviral mechanisms. Indeed, multiple viruses encode specific factors that allow them to directly antagonize host antiviral proteins. The identification and characterization of such viral antagonists is thus highl relevant for understanding human susceptibility to viruses and designing new therapies and vaccines. Despite this great biomedical importance, identifying novel viral antagonists or even which host genes are antagonized by viruses has been difficult. Our previous evolution-guided functional studies have revealed that known viral antagonism of a host gene is often correlated with rapid adaptive evolution, or positive selection, in that host gene. This application seeks to greatly expand the utility of this concept by proposing that positive selection analyses can be used to predict which host antiviral proteins are targeted by unknown viral antagonists and enable identification of the previously unknown antagonists. We will focus on the IFIT (Interferon-induced with tetratricopeptide repeat) gene family, a highly interferon-induced set of broadly-acting antiviral factors that are important for host defense against several viruses. IFIT genes have no known viral antagonists. However several of them have evolved under strong positive selection, leading us to hypothesize that viruses have repeated targeted them with antagonists. We will test whether IFIT proteins are direct targets of viral antagonism by employing a combination of insights gleaned from evolutionary genetics, together with biochemistry and virology techniques. These studies will define the biochemical and functional interactions between IFIT proteins and viral antagonists and test the importance of IFIT antagonism for viral replication. Additional experiments will examine the functional consequences of IFIT evolution, and whether this defines the susceptibility of IFIT proteins to antagonism by currently circulating viruses. Finally, experimental evolution will reveal the evolutionary path that viruses take to counteract IFIT-mediated host defenses. These studies will not only shed light on the role that antagonists play in viral evasion of the IFIT- mediated antiviral response, but will also represent a potentially widely applicable strategy for the de nov identification and characterization of viral antagonists of important antiviral genes.