PROFESSOR, EPPLEY INSTITUTE
Department of Biochemistry and Molecular Biology
Department of Biology and Microbiology
Ph.D. - SUNY StonyBrook, 1974
- 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 in this laboratory involves multiple approaches to studying fundamental problems in stem cell biology and gene regulation. Currently, our research focuses heavily on the roles played by Sox proteins: 1) in controlling the fate of embryonic stem cells, 2) during the reprogramming of somatic cells to a pluripotent stem cell state (iPS cells), 3) in controlling the self-renewal of tumor cells, in particular brain tumor cells, and 4) in the regulation of gene expression, including its own gene, at the transcriptional level. Overall, our stem cell projects seek to shed light on the molecular mechanisms that control the self-renewal of stem cells - a problem that is central to developmental biology, regenerative medicine, and cancer biology.
Expression of Sox2 is regulated within narrow limits in embryonic stem cells: Several studies have identified large sets of genes in embryonic stem (ES) cells that are associated with the transcription factors Sox2 and Oct4. These two transcription factors are part of an essential gene regulatory network responsible for maintaining the properties of ES cells. Not surprisingly, knockdown of either Sox2 or Oct4 leads to death of the early embryo, or, in the case of ES cells, differentiation and loss of self-renewal. Other studies have shown that Sox2 and Oct4 work together as master regulators to cooperatively stimulate the transcription of their own genes, as well as the transcription of a larger network of genes required for embryogenesis. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes (Figure 1), relatively little is known about the mechanisms that prevent their overexpression.
Our studies have shown that elevating Sox2 levels initiates a negative feedback loop (Figure 2), which reduces the endogenous expression of at least five Sox2:Oct4 target genes in ES cells, including the endogenous Sox2 gene itself (Boer et al., Nucleic Acids Research, 35: 1773-1786, 2007).
Moreover, these studies have shown that the ability of Sox2 to limit transcription is dependent on the binding sites for Sox2 and Oct4. 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 shown that a small increase in the level of Sox2 (2-fold or less) rapidly triggers the differentiation of ES cells (Kopp et al., Stem Cells, 26:903-911, 2008). 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 (Figure 3). Moreover, our studies provide new insights into the diversity of mechanisms that control Sox2:Oct4 target genes.
Sox2 associates with a large set of nuclear proteins in ES cells: Although it is clear from our studies that small increases in the levels of Sox2 in ES cells trigger their differentiation, the mechanisms by which Sox2 controls the fate of ES cells are poorly understood. To begin to understand the mechanisms involved, we recently performed an unbiased screen of nuclear proteins that associate with Sox2. For this purpose, we employed the highly sensitive proteomic method known as Multidimensional Protein Identification Technology (MudPIT). These studies demonstrated that Sox2 associates with >60 nuclear proteins (Mallanna, Ormsbee et al., Stem Cells, in press, 2010). Gene ontology analysis of the Sox2-associated proteins indicates that these proteins participate in a wide range of cellular processes. Equally important, a significant number of the Sox2-associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4 and Essrb. Thus, it is evident that Sox2 and other ES cell master regulators are part of a large interconnected protein-protein interaction landscape that controls the fate of ES cells (Figure 4).
Elevating the levels of Sox2 induces the expression of specific bivalent genes: The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in ES cells, yet poised for rapid activation, due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. To explore the regulation of bivalent genes, we took advantage of the fact that elevating Sox2 in ES cells not only induces their differentiation, but also rapidly (within 3 hrs) activates the bivalent gene, Sox21. For these studies, we employed chromatin immunoprecipitation (ChIP) and demonstrated that in ES cells the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation of the Sox21 gene, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Importantly, we determined that none of these changes occur when ES cells differentiate in response to retinoic acid. Moreover, we determined that ES cells lacking a functional PRC2 complex, which is associated with gene silencing in general, fail to activate the Sox21 gene, apparently due to its association with other repressive complexes. Together, our findings argue strongly that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which only exits when appropriate differentiation signals appear. This study was recently published in FASEB Journal, 2010.
E-Mail: Angie Rizzino