Principal Investigators: GREALLY, J.M., LEVY, M.Project abstractIN VIVO IMAGING OF X INACTIVATION. We propose to develop a system for in vivo imaging of the epigenetic regulatory processes involved in Xchromosome inactivation. X inactivation is a well-studied paradigm of epigenetic gene regulation, involving thesilencing of the majority of the genes on one X chromosome in female cells, part of the process of dosagecompensation in mammals. A number of epigenetic regulatory processes have been found to contribute to theinactivation process, which when imaged using immunofluorescence on fixed cells generate a signalthroughout the chromosome territory of the inactive X. The robustness of this signal makes X inactivation anattractive system for the development of in vivo imaging approaches. The inactive X is characterized by thepresence of repressive post-translational histone modifications such as histone H3 lysine 9 trimethylation(H3K9me3) and H3K27me3, modifications established by polycomb group proteins which, when mutated, areassociated with the failure of X inactivation. There are, however, other regulatory mediators implicated withfunctions that are less obviously related to the establishment of these chromatin states, functions such ashelicase activity, RNA-binding, matrix-attachment region DNA-binding, or those functions associated withchromosomal structural maintenance motifs. As a means of understanding how each component of the Xinactivation system interacts functionally, an in vivo system would allow the observation of sequentiallocalization of the protein mediators and histone modifications to the inactivating X chromosome, thusestablishing a likely hierarchy of regulation in this complex epigenetic process. In order to develop such a system, a number of areas of expertise need to be assembled. The projectstarts with the in vitro generation of histone peptides (and eventually entire reconstituted nucleosomes) withmethylation and ubiquitination marks (David Allis and Tom Muir, Rockefeller University) that are then used forin vitro selection by co-PI Matthew Levy (Einstein) to create RNA aptamers specifically binding to these post-translational modifications. These aptamers are then linked in an expression construct to RNA hairpins boundby fluorescently-tagged phage coat proteins, a system pioneered by co-investigator Robert Singer (Einstein) asa means of tracking RNA in vivo in transcription studies. This project represents the first use of the samesystem for epigenetic studies. The cell type in which the system will be optimized will be a female mouseembryonic stem cell line, allowing not only X inactivation studies but also the broader use of this system inpluripotent cells when made available to the scientific community. The X inactivation studies will be facilitatedby the development of fluorescent tags for the candidate protein mediators of X inactivation (Edith Heard,Institut Curie, Paris, France). The project is thus based on a strong and multifaceted foundation of expertiseand resources.PHS 398/2590 (Rev. 11/07) Continuation Format Page
Principal Investigators: GREALLY; J.M.; LEVY; MatthewProject narrativeIN VIVO IMAGING OF X INACTIVATION. In this project; we propose to develop a system that will allow us to see within the cell nucleus howprocesses that are important for switching genes on and off are physically interacting. We are using adramatic example of gene regulation; the inactivation of one of the X chromosomes in female cells. Wealready know that there are numerous proteins that are involved with inactivation of the X chromosome; andwe recognize that the inactive X is marked by the addition of certain chemical groups to the proteins containedwithin that chromosome. What is not apparent is how each of these regulatory mediators controls or iscontrolled by the others involved. At present; we need to kill cells to see where these regulatory mediators arelocated within the cell; but if we were able to watch them in living cells we could determine the order in whichthese regulators exert their effects; thus getting an indication of the regulatory mechanism in this process. We propose to combine a number of technologies that have not been brought together previously in orderto be able to watch how these regulators interact in the living cell. We will use mouse embryonic stem cells; acell type that can turn into most cell types in the body; so the resources we develop will be more generallyuseful to the scientific community and will not be restricted to the study of X inactivation. We will also use anew technology involving nucleic acid structures called aptamers; selecting them for their ability to bindspecifically to the chemical groups or the proteins of interest. The investigators involved represent leaders inthe fields of X chromosome inactivation; chromatin biology; live cell imaging and aptamer technology; and thegoal is not only to gain insights into X chromosome inactivation but also to develop resources that can be usedby the broader scientific community.PHS 398/2590 (Rev. 11/07) Continuation Format Page
| Greally, John M (2017) Population Epigenetics. Curr Opin Syst Biol 1:84-89 |
| Vijayakanthi, Nandini; Greally, John M; Rastogi, Deepa (2016) Pediatric Obesity-Related Asthma: The Role of Metabolic Dysregulation. Pediatrics 137: |
