Daniel J. McKay - US grants

DESCRIPTION (provided by applicant): The organization of the genome into chromatin plays a central role in the regulation of developmental gene expression programs. One widely held belief to explain this regulation is the coordinated control of chromatin accessibility. However, there is little high resolution data measuring chromatin accessibility in vivo, the regulators of large scale chromatin accessibility are largely unknown, and the molecular mechanisms by which chromatin accessibility affects gene activity are unclear. This proposal describes a genomics approach to directly measure chromatin accessibility at high resolution in order to develop a system in the experimentally tractable Drosophila model that will examine the relationship between chromatin accessibility and gene activity. To accomplish this objective, we will utilize a technique developed by the Lieb lab termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). We present data here not only demonstrating the ability of FAIRE to reproducibly isolate discrete regions of 'open'chromatin, but also to identify broad chromosomal regions that share chromatin accessibility properties, even using small numbers of cells. Specifically, we will first perform a genomics screen to identify novel regulators of chromatin accessibility by using RNAi-mediated knockdown in Drosophila S2 cells, followed by analysis of FAIRE-enriched DNA on microarrays. Second, we will generate genomic chromatin accessibility maps for different tissues and stages of Drosophila development by performing FAIRE followed by high-throughput sequencing, providing a framework to assess the relationship between chromatin accessibility and gene activity during development and identifying cis-regulatory modules that control developmental expression programs. Finally, we will begin to characterize potential regulators of chromatin accessibility in vivo through mutational analyses. Relevance to Public Health: Many developmental pathways and mechanisms, when misregulated, contribute to human disease. Therefore, the knowledge gained by these efforts will have applications in understanding mechanisms that underlie disease states such as cancer.

Project Summary/Abstract My lab is broadly focused on developmental gene regulation. We study the transcriptional mechanisms that control determination and maintenance of cell fates over time. In particular, we seek to understand how access to regulatory DNA is spatiotemporally controlled. Because many developmental disorders and diseases acquired later in life are a consequence of gene regulatory defects, a better understanding of the underlying mechanisms is crucial for preventing diseases and improving their outcomes. We combine genomic, genetic and biochemical approaches in Drosophila melanogaster. The fly system offers multiple strengths, including a powerful ability to manipulate gene activity with temporal and spatial precision, small genome size, which affords cheap genomics, and a deep knowledge of the relevant genetic pathways, nearly all of which are conserved in humans. Research during the term of this grant will explore multiple questions at the core of modern developmental biology and the field of epigenetic gene regulation. We will investigate how the transcriptional programs underlying tissue identity are deployed in the proper temporal sequence during development. We have uncovered a temporal cascade of transcription factors which we hypothesize control the sequential activation and inactivation of transcriptional enhancers over time. We have termed these transcription factors as ?chromatin gatekeepers? due to their requirement for opening and closing access to DNA regulatory elements. The mechanisms by which enhancers are inactivated, or ?decommissioned? over time are particularly understudied. One of our objectives is to decipher these mechanisms. We will also investigate how information about decisions made earlier in development is propagated over time. A key to unlocking this question is a unique genetic resource we recently generated that enables us to directly test the function of histones. Histones are subject to a diverse array of post-translational modifications (PTMs) that are thought to serve as carriers of epigenetic information to regulate many DNA-templated processes. However, evidence supporting a functional role of histone PTMs in animals is largely correlative due to the difficulty in creating mutant histone genotypes in animals. Drosophila is distinct amongst animal models in that the histone genes reside at a single locus in the genome. We can replace the endogenous histone genes with tailor-made versions, thereby providing us with the first opportunity to distinguish between regulatory information that is directly encoded in the DNA sequence, and information that is epigenetically propagated. We will employ this approach to interrogate the molecular role that histone PTMs play in enhancer regulation and in heritable control of gene expression. MIRA funding would unify our research topics into a single funding mechanism. This will enable me to spend more time at the bench mentoring students, collaborating with other scientists, and exploring new and unexpected areas of research. Thus, MIRA support would maximize our ability to contribute to a mechanistic understanding of gene regulation, and the in the longer term, to leverage this insight toward understanding of disease etiology and treatment.