In eukaryotic cells, DNA interacts with histone proteins to form a structure called chromatin, the basic unit of which is the nucleosome. While chromatin was once thought to be inert, present only to organize and compact the DNA, it is now clear that chromatin can actively control gene expression. Formation and maintenance of the correct chromatin structure is therefore critical. The goal of this project is to use the powerful molecular and genetic tools of Drosophila melanogaster (the fruit fly) to understand how the structure of chromatin is maintained during gene expression. The research focuses on the role of the chromatin remodeling factor CHD1. CHD1 (chromodomain, helicase, DNA-binding domain protein) is a highly conserved ATPase that is localized to transcriptionally active genes. This project addresses the specific hypothesis that CHD1 is targeted to active genes to promote chromatin reassembly in the wake of transcription, thereby modulating global chromosome structure. This hypothesis will be tested through two specific aims: (1) Investigate changes in chromatin composition and histone modifications following loss or gain of CHD1, and (2) Identify proteins that functionally interact with CHD1 using a biochemical approach complemented with a novel genetic assay. To address these aims the investigator will employ a variety of genetic, biochemical, and cell biological tools and techniques that are ideal for the investigation of chromosome morphology.
Broader impacts: The understanding of how chromosome structure is maintained during gene expression is relevant to our basic understanding of cell biology. A more immediate impact of this research is the involvement of undergraduates. Undergraduate students from Claremont McKenna, Scripps, and Pitzer Colleges of the Claremont Colleges will carry out much of this research under the mentorship of the investigator. These three top-tier liberal arts institutions share the investigator's unique department (The Joint Science Department, consisting of physicists, chemists, and biologists). Most of the science majors conduct research as part of a senior thesis, and many students begin research in their sophomore or junior years. Over the three years of this project, the investigator will provide a substantial (often multi-year) research experience for 10-15 undergraduates, providing them with skills to help them succeed in postgraduate education and in their chosen careers. This will allow undergraduates to have true ownership of their own projects, and participate in research at a level usually reserved for graduate students. Senior level undergraduates will have the opportunity to participate in the training of younger undergraduates, giving them insights into how to teach science and explain how research is done.
This project used the powerful genetic system of Drosophila melanogaster (the fruit fly) to understand how chromosome structure is maintained. As the proteins involved in these processes are highly conserved, findings are applicable to other organisms, including humans. A huge international "fly community" has generated a wealth of tools and techniques, making Drosophila a prominent and well-accepted model organism. In eukaryotic cells, DNA interacts with histone proteins to form a structure called chromatin, the basic unit of which is the nucleosome. While chromatin was once thought to be inert—present only to organize and compact the DNA—it is now clear that chromatin is not passive; rather it can actively control gene expression. This project focused on a protein that is conserved in yeast, flies, and humans called CHD1 (named for its protein domains: chromodomains, helicase, DNA binding domain). The Intellectual merit portion of this project focused on two specific aims. Aim 1. Investigate changes in chromatin composition and histone modifications following loss or gain of CHD1. One of the outstanding features of Drosophila is their giant polytene chromosomes. We found that either loss of CHD1 or over-expression of CHD1 results in the disruption of normal chromosome structure. We investigated these exciting and unusual chromosome phenotypes by using immuno-fluorescence to analyze the levels and distributions of histone modifications, histone variants and non-histone proteins. Our studies revealed that the CHD1 protein functions as a rheostat; either too much or too little has consequences for chromatin structure. Surprisingly, changes in levels of CHD1 had little effect on histone marks enriched at active genes, while CHD1 levels appear important for the maintenance of transcriptionally inactive chromatin. This work was recently published with student co-authors [Bugga, L., McDaniel, I.E., Engie, L., and Armstrong, J.A. (2013). The Drosophila melanogaster CHD1 chromatin remodeling factor modulates global chromosome structure and counteracts HP1a and H3K9me2. PloS ONE 8, e59496.] This project also focused on the role of CHD1 in histone deposition during gene expression. Our studies revealed that loss of CHD1 had consequences for chromatin assembly at active genes. These findings were published as part of a large collaboration with several groups who addressed the same question in yeast [Radman-Livaja, M., Quan, T.K., Valenzuela, L., Armstrong, J.A., van Welsem, T., Kim, T., Lee, L.J., Buratowski, S., van Leeuwen, F., Rando, O.J., et al. (2012). A key role for chd1 in histone h3 dynamics at the 3' ends of long genes in yeast. PLoS genetics 8, e1002811.] Aim 2. Identify proteins that functionally interact with CHD1. Much can be learned about the function of a protein by identifying its protein partners. We used immunoprecipitation and mass spectrometry to identify proteins that physically associate with CHD1 in the Drosophila embryo. To complement this biochemical approach, we devised a wing-based genetic assay to screen candidate genes for interactions with chd1. Together these approaches together allowed us to identify a short list of putative CHD1 interaction partners. Broader impacts: This project resulted in the training of 24 undergraduate students from Claremont McKenna, Scripps, and Pitzer Colleges (which together share the W.M. Keck Science Department of the Claremont Colleges); three undergraduate students from other institutions; and two high school students. Several Claremont College students presented their research at Annual Drosophila Research Conferences, and three undergraduates co-authored publications. A Pitzer undergraduate wrote a Matlab program with a graphic user interface (GUI) that allows us to quantify immunofluorescence from polytene chromosomes while automatically creating a mask over the chromosomes to easily subtract any background signal. This program is broadly applicable and has been used by investigators in other fields.
