Identification and Analysis of Genome-wide Intra- and Inter-chromosomal Associations in Human Embryonic Stem Cells
We used to think of genes as discrete segments of DNA that code for proteins and that are regulated or controlled by nearby or adjacent DNA sequences. Recently, it has been shown that DNA sequences on the same chromosome that are very distant from a gene may regulate its function, determining if the gene will be turned on or turned off. We have now discovered that DNA sequences that are even on different chromosomes from the protein-coding gene may control this gene. These interactions are often totally unexpected. For example, the NF1 gene, which causes neurofibromatosis, a genetic disease that may lead to the development of brain tumors, lies on chromosome 11 in the mouse, but the production of its protein product is partially under the control of a DNA segment near a growth factor gene(IGF2) on mouse chromosome 7; the same relationship appears to hold true in humans. No one had ever suspected such an interaction, and it is now logical to consider targeting the IGF2 gene as part of our hopes to treat neurofibromatosis. In other words, the discovery of a long-range interacting sequence of DNA provides us with totally new targets for drug development. Several other groups have also shown that inter-chromosomal gene regulation can control immune genes and the genes that regulate our sense of smell. We believe that many, if not most, genes are controlled, as least in part, by long range inter-chromosomal interactions, and we have proposed a new method to try to define all of these interactions in cultured human cells. It is highly likely that many of these long range inter-chromosomal interactions are important in the regulation of a gene, and therefore, we plan to catalog all of these interactions in human embryonic stem cells, and see whether the interactions remain the same or are altered as the cells differentiate into fat cells, and, in the future, into other kinds of cells. This catalog will provide us with numerous new clues to the regulation of disease-related genes, and will suggest new drug or gene-therapy related targets to treat these diseases. By comparing the list of interacting genes in normal embryonic cells with those in cells harboring a genetic disease, we may develop new diagnostic tools in addition to potential therapeutic avenues.
Statement of Benefit to California:
Our goal is to characterize how genes are regulated by distant DNA segments on different chromosomes. We will develop a complete catalog of these long range interactions, which will provide a list of how one gene may regulate another. This catalog will provide the framework for determining how disease-related genes are controlled and will allow investigators to develop new diagnostic tools and/or new therapeutic targets to treat various genetic diseases. These are targets that had never heretofore been considered because the long range interactions among these genes had not been known. Clinical translation of these studies into new drugs will be of great benefit to the people of California.
SYNOPSIS: The goal of these studies is to explore long-range DNA interactions as they occur on chromosomes of hES cells and their differentiated adipocyte progeny. The PI proposes to use novel techniques called chromosome confirmation capture (3C) and associated chromosome trap (ACT, developed in the PI's laboratory) to identify all of the long-range intra and inter-chromosome interactions in hES cells. The idea is to eventually generate a set of microarrays which will contain hybrid sequences that will read-out the interactions present in a given cell sample. The rationale for these studies is the well-documented role of long-range interaction in the control of gene expression. The availability of such a microarray base reagent would be of considerable value. The PI proposes to use two hES cell lines derived by the Wisconsin group. SIGNIFICANCE AND INNOVATION: The major strength of this proposal is the development and application of very new technologies to elucidate the repertoire of long-range intra and inter-chromosomal interactions in hES cells. Such interactions have been shown to play key roles in gene regulation in several systems. It is likely that similar interactions will be important in hES cells, and therefore, efforts to identify and catalog these are significant. STRENGTHS: The major strength of the proposed studies is the application of very new technologies to ask highly novel questions in the hES cell system. It is likely that both the 3C and the ACT techniques will be applicable to hES cells. The PI has ample experience with these technniques in other cell types. Moreover, the PI's laboratory has recently published a paper that describes an inter-chromosomal interaction between the Igf2/H19 imprinting control region on mouse chromosome 7 and a region of chromosome 11. This interaction controls the expression of the NF1 and Wsb1 genes (chromosome 11). The possibility that the proposed studies will generate a catalog of most, if not all long-range interaction present in hES cells and their differentiated progeny is exciting. In particular, if the studies yield microarray reagents that can easily interrogate cell populations for such interactions. The PI has the necessary expertise for these studies, and in general they are well-described. WEAKNESSES: The major weakness in the proposed studies is that the 3C and the ACT technologies are very complex, and as presented, require a considerable effort to comprehend. Given the expertise of the PI, this may not be a formidable drawback. However, a more detailed description would certainly have been appropriate and appreciated. Additionally, it would have been reassuring if at least some preliminary proof-of-principle data demonstrating the applicability of these technologies in hES cells were included. No evidence is presented to indicate that the ACT assay described by the PI actually works. Indeed the assay is impossibly complicated. Also there is no description of how the final dataset will be used to learn any biology and how false discoveries will be weeded out. Except to be technically responsive to this RFA, it is not clear why this assay would, should it work, be suitable for ESC research. DISCUSSION: A reviewer believes that both the 3C and ACT technologies will be applicable to hESC, and that the PI has ample experience in other cell types. Another reviewer feels that 3C is feasible but that ACT is impossibly complicated and that there is no evidence that it would be a successful assay. In addition, mapping all of the interactions and building microarrays is on the cutting edge itself, and therefore is high risk. There was discussion over whether the 3C assay alone would be valid, and whether it would be worthwhile to start a process for an "all or none" technology. What would be deemed false positives or false negatives, and how would these be sorted out? A reviewer was not convinced that the studies would result in anything but useless noise.