Professor, Eppley Institute

Department of Biochemistry and Molecular Biology,
Department of Biology and Microbiology

Ph.D. - SUNY StonyBrook, 1974

Awards:

▪ Distinguished Teaching Award

▪ Outstanding Faculty Mentor of Graduate Students Award

▪ UNMC Distinguished Scientist Award

Editorial Review Boards:

▪ International Journal of Developmental Biology - Associate Editor for the Americas

▪ Molecular Reproduction and Development - Associate Editor

▪ In Vitro Cellular and Developmental Biology - Associate Editor

▪ Cytotechnology - Editorial Board

Research Interests

Research in this laboratory focuses on two fundamental problems in gene regulation and stem cell biology. The first deals with the molecular mechanisms responsible for regulating gene regulatory networks that control the self-renewal of stem cells. The second deals with molecular mechanisms that control the selective binding of transcription factors to gene regulatory sequences. Details for each project are discussed below to help explain the long-term goals of this laboratory. Understanding gene regulatory networks that coordinate the transcription of sets of genes during cell growth and differentiation is one of the biggest challenges facing biomedical scientists. Deciphering the mechanisms that control gene regulatory networks at the molecular level will not only enable us to better understand normal physiology, it will also provide critical insights into aberrant gene regulation in diseases, such as cancer. Although substantial progress has been made in understanding the mechanisms used to regulate individual genes, relatively little is known about the molecular mechanisms used to regulate the transcription of sets of genes (coordinate gene regulation). A major goal of the work in this laboratory is to understand the fundamental mechanisms and signaling pathways that orchestrate the early stages of mammalian embryogenesis and control the self-renewal and pluripotency of embryonic stem (ES) cells.  Currently, our studies focus on the molecular mechanisms that control the self-renewal of stem cells, because this problem is central to our understanding of developmental biology, regenerative medicine and cancer biology.  In the case of cancer, learning how to disrupt the self-renewal of cancer stem cells holds the key to more effective cures in cancer.  To better understand the self-renewal of stem cells, we are studying a gene regulatory network used to coordinately control the transcription of genes that play key roles in the self-renewal of ES cells. As part of these studies, we are are examining how ES cells coordinately control the transcription of six of their genes (FGF-4, UTF1, Sox2, Oct-3/4, Fbx-15, and Nanog) that are regulated by the cooperative and synergistic action of the transcription factors Sox2 and Oct-3/4 (also known as Oct4). For each of these genes, Sox2 and Oct-3/4 binds cooperatively to two closely-spaced DNA regulatory elements that exert powerful effects on the promoters of each of the six genes (Figure 1)  Disruption of the two DNA regulatory elements in each gene leads to a dramatic decrease (>90%) in the activity of each gene’s promoter.  ES cells are employed in our studies, because they serve as excellent model systems for early mammalian development and because differentiation of these cells suppresses their ability to form tumors. Thus, this model system provides an excellent opportunity to study the molecular mechanisms by which differentiation blocks the malignant properties of tumor cells. For our studies, a wide range of approaches are employed, including: gene cloning, gene targeting, DNA arrays, construction of promoter/reporter gene constructs, PCR, site-directed mutagenesis, gel mobility shift analysis, in vitro footprint analysis, western, northern and Southern blot analyses, in vitro transcription/translation, and chromatin immunoprecipitation (ChIP). Recent and Current Work: Several studies have identified large sets of genes in embryonic stem (ES) cells that are associated with the transcription factors Sox2 and Oct-3/4.  Other studies have shown that Sox2 and Oct-3/4 work together as master regulators to cooperatively stimulate the transcription of their own genes as well as a larger network of genes required for embryogenesis.  Moreover, small changes in the levels of Sox2:Oct-3/4 target genes, such as Oct-3/4 and Sox2, alter the fate of stem cells.  Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, relatively little is known about the mechanisms that prevent their overexpression.  Our studies have shown that elevating Sox2 levels initiates a negative feedback loop, which reduces the endogenous expression of at least five Sox2:Oct-3/4 target genes in EC cells and ES cells (Boer et al. Nucleic Acids Research, 35: 1773-1786, 2007).  Moreover, these studies have shown the ability of Sox2 to limit transcription is dependent on the binding sites for Sox2 and Oct-3/4.  In addition, this effect of Sox2 is dependent on its transactivation domain, which is located at its C-terminus.    These studies led to the prediction that elevating the levels of Sox2 in ES cells would induce their differentiation.  To test this hypothesis, we engineered mouse ES cells for inducible overexpression of Sox2.  Using this model system, we have recently shown that a small increase in the level of Sox2 (2-fold or less) rapidly triggers the differentiation of ES cell (Kopp et al., Stem Cells, 26:903-911, 2008). Together, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes.  Importantly, our studies argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network that orchestrates mammalian embryogenesis as well as the self-renewal and pluripotency of ES cells.  As part of our ongoing studies, we are testing our hypothesis that nature uses a squelching mechanism, as part of a negative feedback loop, to help maintain the proper expression of Sox2:Oct-3/4 target genes during development.  We are also examining whether genes activated rapidly by Sox2 overexpression play an critical role in the induction of ES cell differentiation. Research in this laboratory also focuses on transcription factors that belong to the Ets superfamily. Our current efforts seek to understand how Ets proteins regulate genes that play key roles in cancer. For these studies, we are focusing on the Ets proteins, Ets1, Ets2 and Elf3, and a target gene of Ets proteins, the type II TGF-β-receptor gene (Kim et al., JBC 277:17520-17530, 2002; Kopp et al., JBC 279:19407-19420, 2004). The type II TGF-β-receptor gene is of interest, because it mediates the effects of the multi-functional factor TGF-β, which exerts both positive and negative effects on tumor growth and metastasis in many cancers.  Ets proteins are of interest because Ets proteins are overexpressed in many cancers.  Several years ago, our laboratory demonstrated that Elf3, like seven other Ets proteins, contain an autoinhibitory domain, which regulates its binding to DNA. To better understand the molecular mechanisms by which Elf3 regulates these processes, we created a large series of Elf3 mutant proteins with specific domains deleted or targeted by point mutations.  The modified forms of Elf3 were used to analyze the contribution of each domain to DNA binding and the activation of gene expression.  Our work demonstrates that three regions of Elf3, in addition to its DNA binding domain (ETS domain), influence Elf3 binding to DNA, including its transactivation domain, which behaves as an autoinhibitory domain.  Importantly, disruption of the transactivation domain relieves the autoinhibition of Elf3 and enhances Elf3 binding to DNA (Kopp et al. JBC 282:3027-3041, 2007).  Moreover, we have shown that the N-terminal region of Elf3, which contains the transactivation domain, interacts with its C-terminus, which contains the ETS domain.  Equally important, we have shown that the interaction between the two domains of Elf3 is disrupted when the transactivation domain of Elf3 interacts with the co-activator Med 23.  Other work in this laboratory indicates that this unexpected role of transactivation domains is not limited to Ets proteins.  We have shown that the transactivation domain of the transcription factors Sox11 behaves as an autoinhibitory domain (Wiebe et al., JBC 278:17901-17911, 2003).  Together, our studies lead to a new paradigm for gene activation and the mechanisms by which transcription factors are selectively recruited to the correct set of genes in a given cell type.   More specifically, our findings suggest a model in which the binding of some, if not most, transcription factors to DNA is mediated by first interacting with an appropriate co-activator that converts them from an autoinhibited form to a DNA binding form.   Currently, it is commonly argued that, after transcription factors bind to DNA, they recruit co-activators to the gene.  Our model posits that transcription factors, such as Ets proteins, must interact with appropriate co-activators in order to bind to DNA.  Accordingly, our model predicts that co-activators play a key role in determining which sets of genes are activated by transcription factors whose transactivation domains function as autoinhibitory domains.

Dr. Rizzino’s laboratory has also used DNA arrays to monitor the expression of genes in embryonic stem (ES) cells and their differentiated counterparts (Kelly, D.L. and Rizzino, A. DNA Microarray Analyses of Genes Regulated During the Differentiation of Embryonic Stem Cells. Molecular Reproduction and Development, 56:113-123, 2000). ES cells are derived from the inner cell mass of blastocysts and, in response to retinoic acid (RA), are induced to differentiate to form some of the first distinguishable cell types of early mammalian development. Additionally, ES cells are of significant interest to those characterizing gene function by gene targeting (Wilder, P. and Rizzino, A. Mouse genetics in the 21st century: Generating a cornucopia of mouse mutants by gene targeting. Cytotechnology, 11:79-99, 1993.). With the advent of DNA microarray technology, which allows for the study of expression patterns of a large number of genes simultaneously within a cell type, there is an efficient means of gaining critical insights to the expression, regulation and function of genes involved in mammalian development for which information is not currently available.

Attached below are four files (Microsoft Excel) that contain lists of genes that are: 1) expressed at the RNA level by ES cells (Table 1); 2) not detected at the RNA level in ES cells (Table 2); 3) expressed at the RNA level by ES-differentiated cells (Table 3); and 4) not detected at the RNA level in ES-differentiated cells (Table 4). In the case of ES-differentiated cells, differentiation was induced by the addition of retinoic acid to serum-containing medium supplemented with LIF (Kelly and Rizzino, Molecular Reproduction and Development, 56:113-123, 2000). Under these conditions, ES cells differentiate into only one or a few cell types, which exhibit the properties of parietal extra-embryonic endoderm. The genes expressed by ES cells or their differentiated counterparts are listed in rank order based on average pixel intensity. Genes that are up-regulated or down-regulated when ES cells undergo differentiation are contained in Tables 1 and 2 (Molecular Reproduction and Development, 56:113-123, 2000).

Table 1
Table 2
Table 3
Table 4

Publications

Phone: (402) 559-6338

Fax: (402) 559-4651

E-Mail: Angie Rizzino