To address the research goals of this project, Dr. Telfer's laboratory will study how the protein Runx3 regulates expression of an important gene in T cells called CD4. The CD4 gene encodes a protein on the surface of T cells that helps determine their function. Runx3 binds to the DNA containing the CD4 gene during T cell development, and in some of the developing T cells, catalyzes the permanent silencing of CD4 expression. This silencing is inherited; persisting even after the cell divides multiple times as a long-lived mature T cell in the blood. Since this gene silencing is so persistent, it is probable that it is the result of a Runx3-mediated modification of the DNA-associated proteins known as histones. All of the DNA in a cell is wrapped around multiple groups of histones, like a long thread on many spools, in order to package the DNA compactly enough to fit into the nucleus. Modification of histones is known to regulate the expression of genes by regulating how loosely or tightly the DNA is packaged. Genes located in tightly packaged DNA are not expressed and this tightness of packaging and resulting silencing of genes is inherited by the daughter cells. Dr. Telfer's laboratory will investigate whether Runx3 silences CD4 expression in immature T cells by attracting histone-modifying enzymes to the DNA containing the CD4 gene. These enzymes could then modify the histones so that the DNA containing the CD4 gene would be packaged more tightly and expression of CD4 silenced. Introducing mutated or normal forms of Runx3 into primary immature T cells and examining their effects on proteins such as histones associated with the CD4 gene will test this hypothesis. Understanding how permanent gene silencing is mediated is very important for understanding processes in cells such as stem cell self-renewal and differentiation. The research and educational goals of this project are highly integrated with the educational goals of the Department of Veterinary and Animal Science and the Institute for Cellular Engineering (ICE) at the University of Massachusetts Amherst. ICE represents a research and educational collaboration between biologists pursuing questions regarding the mechanisms of stem cell differentiation into specific cells and engineers developing processes to produce stem cells or differentiated cells reliably and in bulk. In this context, Dr. Telfer will improve the teaching of undergraduates in an introductory cellular and molecular biology course through the implementation of methods designed to encourage active learning by the students and rapid assessment of the student's knowledge acquisition by the teacher. These new methods represent a paradigm shift from the traditional teaching method of lecture and exams. The aim of active learning and rapid student assessment is to teach deep learning techniques, which are critical to the retention of a knowledge base. The success of these methods will be assessed by midterm and end-of-semester student evaluation forms as well as by interviews with faculty members teaching the same students in subsequent years. The retention in science of female and minority students will be improved through more effective teaching and by encouraging undergraduate and graduate research experiences in this research project and other related research projects under the collaborative umbrella of ICE at the University of Massachusetts Amherst.
DNA, and especially the minority of DNA that contains genes, is the instruction manual and toolbox for every cell. The control of which genes are copied at what times in the life of a cell is critical, and is often determined by factors outside of the DNA sequence itself, known as "epigenetic" factors. The DNA of each chromosome is a long thread-like molecule that is organized and protected from breakage or tangling by wrapping around spool-like nucleosomes, each one of which is made up of eight histone proteins. The nucleosomes can be spaced far apart and the DNA-nucleosomes coiled loosely for the copying of genes. If the nucleosomes are spaced closely and the DNA-nucleosomes are coiled tightly, genes are blocked from being copied. The tightness of packaging can be controlled by the modification of the histone proteins in the nucleosomes. Some of the histones (histone H3 and histone H4) have tails that protrude from the nucleosome. These tails are modified with the acetyl or methyl chemical flags by enzymes recruited to that gene via transcription factor proteins that recognize and bind to a specific six nucleotide zip code. When the histones are tagged with acetyl groups, the DNA relaxes and is more accessible to the gene copying proteins. On the other hand, when the histones are tagged with methyl groups in certain places on the histone tails, the DNA coils tightly and is not open to gene copying proteins. Remarkably, the state of DNA is remembered even after cell division, before which all the DNA constricts to the densest state, that of the rod-like chromosome, and then decondenses in the daughter cells to the same looseness or tightness that exited prior to cell division. This ability is very important to preserve cell identity in a multi-cellular organism, since it would not behoove the organism to have a liver cell divide, forget that it was a liver cell, and switch to copying the genes that would make it a muscle or bone or cancer cell. This NSF-funded project addressed the problem of the remembrance of gene copying past by investigating how the transcription factor protein Runx triggers the permanent silencing of the CD4 gene in developing cytotoxic T cells. We had previously shown that this silencing is so permanent that the CD4 gene in mature cytotoxic T cells ignores a truncated Runx mutant protein that revs up CD4 gene copying in immature thymocytes, even though these cells have divided many times since the CD4 gene was initially silenced. To test the hypothesis that the initial Runx-mediated silencing of the CD4 gene resulted from histone tagging, we fused Runx mutants that increased instead of blocking CD4 gene copying, or were not very good at turning CD4 copying off, with histone modifying enzymes and tested the result in immature T cells that had not yet turned off CD4. We expected that the fusion of an enzyme that adds methyl groups to histone H3 tails and is associated with making DNA inaccessible to gene copying proteins would give the Runx mutants the ability to silence CD4. Surprisingly, it had the opposite effect- these fusions led to more copying of the CD4 gene. When we investigated the density of the nucleosomes and the histone tags around the CD4 gene of cells treated with a cell-membrane permeable form of the mutant Runx that leads to more copying of the CD4 gene, we found that the nucleosomes were less dense and thus that that the gene would be more accessible, but that the histone tags were unchanged. In addition, the change in nucleosome density and the accessibility of the gene was not linked to the ability of the Runx mutant to bind to DNA, which is absolutely required for it to increase the copying of the CD4 gene. This indicates that the Runx mutant must be acting to increase CD4 copying, and by extension, full-length Runx must be initiating the silencing of CD4, by an as yet unknown mechanism. This means that the elucidation of unx-mediated CD4 silencing has to the potential to lead to the discovery of a previously unknown mechanism of turning gene copying off. We have also characterized the Runx-mediated repression of the copying of the important gene c-myc. C-myc is different from CD4 in that many cells turn c-myc gene copying on and off; whereas CD4 is permanently silenced in cytotoxic T cells and permanently expressed in helper T cells. Future studies will be required to determine how Runx initiates CD4 silencing and whether this occurs via the same mechanism with c-myc and CD4.
