Study of TBX3 Function in human Embryo Stem (hES) Cell Differentiation and Identification of Genome-Wide TBX3 Promoter Binding-Sites with the CHIP-GLAS Promoter Array
Stem cells can develop into every cell, every tissue and every organ in the human body, e.g. they can make any kind of cells in the human body. Stem cells reproduce themselves many times over and over. Their almost limitless potential has made stem cells a significant focus of medical research. But before scientists can use stem cells for medical purposes, they must first learn how to harness their power. They can't treat disease until they learn how to manipulate stem cells to get them to develop into specific tissues or organs. We would like to be able to grow a particular type of cell in the laboratory and then inject it into a patient, where it would replace diseased tissue. But stem cells are not yet being used to treat disease because we still haven't learned how to direct a stem cell to differentiate into a specific tissue or cell type (brain vs. liver, for example) and to control that differentiation once the cells are injected into a patient. We know that turning genes on and off is crucial to the process of differentiation, so we can add some factor into the culture dish and observe stem cells to differentiate into specific types of cells. But some sort of signal is needed to actually trigger the stem cells to differentiate. We are still searching for that signal. If we can ultimately learn how to direct stem cells to differentiate into one type of tissue or another, they can use them to treat the patients. In this proposal, we will first examine. We propose a novel approach to understanding differentiation of human embryo stem (hES) cells, by studying TBX3, a protein called a transcription factor that controls the expression of other genes. In humans, the loss of function of TBX3 causes Ulnar-Mammary Syndrome, a genetic disorder that can pass from one generation to the next. Our studies show that TBX3 can prevent the aging of mouse cells, so that the cells can keep growing. Furthermore, our preliminary results show that TBX3 is downstream mediator of another protein, BMP4. BMP4 is a known key regulator for hES cell differentiation. Thus, TBX3 is an attractive candidate as a downstream mediator of BMP4 in hES cell differentiation. We will test TBX3 effects on hES cell differentiation if down-regulate TBX3 in hES cells with a technology called siRNA knockdown. We will identify the genes controlled by TBX3 with a recently invented powerful technology called CHIP-GLAS. This technique allows us to examine thousands of genes on a small chip in a single experiment. We expect that the innovative experiments proposed here will open a new avenue to understanding the signal of hES cell differentiation.
Statement of Benefit to California:
In this propose, we will collaborate with AVIVA SYSTEMS BIOLOGY (ASB). ASB is a start-up biotech company in California to develop comprehensive mapping of DNA/Transcription factor interactions on a genomic scale. ChlP-GLAS (chromatin immunoprecipitation-guided ligation and selection) technology is the major product of the company. We have completed the pilot study. The preliminary results are very interesting and demonstrate the feasibility of the assay. ChlP-GLAS was invented by Dr. Xiangdong Fu at the University of California, San Diego (UCSD) and licensed exclusively to ASB. ASB has spent the last two years developing ChlP-GLAS technology. ChlP-GLAS utilizes an oligonucleotide microarray containing unique 40-mer regions of 20,000 human promoters. Corresponding to the 20,000 unique regions, a pool of sequence-specific oligos is allowed to anneal to the ChIP sample DMA. These oligos also contain common overhangs for subsequent amplification and labeling. As a result of this selection process only the DNA sequences of interest are amplified. Nonspecific and repeat-containing elements are removed, greatly increasing the sensitivity and specificity of the assay. Additionally, the amplified DNA molecules are all of uniform size, improving amplification and hybridization conditions. Therefore, ChlP-GLAS technology may represent a new generation of ChlP-on-chip which offers increased sensitivity with much less starting material required. On of the aim is validate application of CHIP-GLAS in stem cell research. As the manufacturer of ChlP-GLAS products and supplier of ChlP-GLAS services, ASB will able to capitalize this invention, which will have a significant benefit for the economy of California State. In addition, California as a largest state in US, the proposal study will benefit many of the patients. The aim of this proposal is ultimately to learn how to direct stem cells to differentiate into different type of tissues or cells, so we can use them for medical purposes. For example, stem cells could also be used to repair cells or tissues that have been damaged by disease or injury. This type of treatment is known as cell-based therapy. One potential application is to inject embryonic stem cells into the tissue Stem cells may also one day be used to repair brain cells in patients with Parkinson's disease. Eventually, we might even be able to grow entire organs in a laboratory to replace ones that have been damaged by disease. Growth factors specific to the organ would be added to guide the organ's development.
SYNOPSIS: The proposal revolves around a single gene, TBX3. Mutation in the TBX3 gene causes “Ulnar Mammary Syndrome”, a disease that manifests itself by lack of mammary glands and limb deformity. TBX3 is a BMP target, and mice knocked-out for TBX3 are embryonic lethal, suggesting an early essential function for the gene. This proposal has two specific aims. The first is to examine TBX3 functions in hES cell differentiation. The second specific aim is to identify the genome-wide downstream targets directly bound by TBX3 in hESC with chromatin immunoprecipitation-guided ligation and selection (CHIP-GLAS). INNOVATION AND SIGNIFICANCE: There are many genes that are the targets of BMPs and necessary for early embryonic development. It is not very clear why this one is more important than the others. It is also not very clear why the resolution of the embryonic function of TBX3 is better addressed in the context of hESCs than in the mESCs. Therefore, while this is an interesting project, it lacks both the higher level of significance and of innovation. STRENGTHS: The proposal is well written and proposes experiments that are well designed, sound and feasible. There is little doubt that the PI will successfully accomplish the aims of his proposal. WEAKNESSES: While there is nothing wrong with the aims proposed, this grant falls short from addressing top priority issues in hESCs. The very straightforward approaches described can easily be applied to NIH registry hES cell lines. This type of proposal could be a focus of an NIH appication, because it is likely that any uncovered role(s) of Tbx3 will be shared by independent lines. DISCUSSION: There was no further discussion following the reviewers' comments.