We are investigating a widespread and highly conserved, yet poorly understood, form of transcriptional control whereby gene expression on a global level is abruptly upregulated in a process termed genome activation. Our system is the developing germline of the nematode C. elegans, and thus we use genetics, genomics, and cytology to study how genome activation is controlled. Previously published and preliminary data from our group have outlined a genetic pathway for germline genome activation, whereby the topoisomerase II (TOP-2) enzyme is activated, in a signal-mediated manner, to produce programmed DNA breaks. The purpose of these breaks is to recruit the TIP60/RUVB regulator to chromatin, so that genome decompaction occurs, thereby facilitating genome activation. In this proposal we study three distinct components of the genome activation pathway that we have discovered. First, we study how genome architecture is established in primordial germ cells (PGCs) prior to activation. Our preliminary data suggest that whole-genome heterochromatization is the means by which this architecture is established and the means by which mRNA transcription is globally repressed in resting PGCs. We are unaware of any other examples in eukaryotic biology where heterochromatin formation is employed on such a grand scale, and thus these experiments are likely to supply a novel paradigm for how gene expression can be globally repressed in resting cells. We will also examine how TOP-2 is activated by signaling to go on and induce DNA breaks in the germline genome. It has been appreciated for some time that programmed DNA breaks occur in the genome during meiosis, however our discovery of programmed breaks occurring much earlier in germline development is unprecedented and worthy of detailed investigation. We plan to study how TOP-2 is regulated and we will also determine where in the genome the breaks are made. Successful completion of these experiments will provide a novel mechanism for programmed break formation that is likely to be relevant across cell types and organisms. A final set of experiments will examine how genome decompaction occurs. Our data show that the sole purpose of TOP-2 induced breaks is to recruit the TIP60/RUVB chromatin regulator to DNA so that decompaction can occur. Why PGCs take such extreme measures -- intentionally inducing a dangerous form of DNA damage into their genomes -- for the purpose of chromatin decompaction is a fascinating question. To get at this we will study how broken DNA recruits TIP60/RUVB and how the complex then decompacts chromatin. We will determine distance thresholds for positioning of engineered DNA breaks and target gene activation. Lastly, we will examine the exciting possibility that gene mobility within the nucleus is coupled to decompaction, and that mobility is important for transcriptional activation.

Public Health Relevance

This proposal examines how the germline genome is activated as germ cells initiate proliferation. We use the genetically amendable nematode C. elegans as a model system to study this problem. Previous work from our laboratory found that during genome activation the DNA is intentionally broken and that these programmed DNA breaks are required for genome expression. In this proposal we take advantage of the sophisticated genetics and cytology afforded by the C. elegans system to study how the genome is structured, architecturally, and how DNA breaks are needed to dismantle this architecture so that germline genes can be activated and germline proliferation may ensue.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Keane-Myers, Andrea
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University of Southern California
Schools of Arts and Sciences
Los Angeles
United States
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