Epigenetic factors participate in the development and organogenesis in higher organisms. For the underlying process of differentiation, the emergence of epigenetic individuality of cells within the organism is required. Our application aims at developing a method for preparing global profiles of histone modification states over whole chromosomes, from single cells. The measurement of epigenetic individuality also becomes important in understanding organs undergoing rapid change, such as cancers that include cancer stem cells. The proposed method is conceptually similar to fiber-FISH. In particular, we plan on extracting chromosomes from a single, targeted cell. We then plan on visualizing the histone modification pattern by stretching whole chromosomes and fluorescent imaging of antibodies coupled to those histone tail modifications. In order to provide homogeneous stretching of ultra-long chromatin molecules, we will utilize a novel stretching technique based on nanofluidic channels. We have recently shown an elongation of 49 kbase chromatin molecules in such a channel to about 3 micrometers, leading to an equivalent expected resolution 6 kbase under fluorescence microscopy. The use of nanochannels enables stretching of ultra-long molecules without the problem of tension- based breaking such as in molecular combing. We propose to demonstrate the utility of our approach by following the global epigenetic state of early C. elegans embryos. Since C. elegans is an organism with an extremely early commitment of some cells to specific cell lines, we anticipate individuality of cells within such embryos. On the other hand, we will also be able to probe whether pluripotent cells are identical, or are also characterized by some individuality. All preliminary work will also be performed on the embryonic C. elegans system. Our application is structured into three distinct parts. Part one investigates whether whole chromosomes derived from mitotic cells can be stretched inside nanochannels. Part two is concerned with the design, development, and testing of a microfluidic platform that enables automated lysis of embryos and cells, and introduction into nanochannels. Part three will follow the epigenetic embryonic development from the 8 to the 28 cell stage.
This project has the potential to improve public health by developing a new technique for monitoring the epigenetic programming state of genomic sized molecules, ideally whole chromosomes, from single cells. Epigenetic programming has broad implications critical to both diseased and healthy states, in particular cancer development. This proposed work will enable tracing the epigenetic individuality of single cells from complex tissues.
|Lim, Shuang Fang; Karpusenko, Alena; Sakon, John J et al. (2011) DNA methylation profiling in nanochannels. Biomicrofluidics 5:34106-341068|