Angie Rizzino

Small Increases in the Level of Sox2 Trigger the Differentiation of Mouse Embryonic Stem Cells

Previous studies have demonstrated that the transcription factor Sox2 is essential during the early stages of development. Furthermore, decreasing the expression of Sox2 severely interferes with the self‐renewal and pluripotency of embryonic stem (ES) cells. Other studies have shown that Sox2, in conjunction with the transcription factor Oct‐3/4, stimulates its own transcription as well as the expression of a growing list of genes (Sox2:Oct‐3/4 target genes) that require the cooperative action of Sox2 and Oct‐3/4. Remarkably, recent studies have shown that overexpression of Sox2 decreases expression of its own gene, as well as four other Sox2:Oct‐3/4 target genes (Oct‐3/4, Nanog, Fgf‐4, and Utf1). This finding led to the prediction that overexpression of Sox2 in ES cells would trigger their differentiation. In the current study, we initially engineered mouse ES cells for inducible overexpression of Sox2. Using this model system, we demonstrate that small increases (twofold or less) in Sox2 protein trigger the differentiation of ES cells into cells that exhibit markers for a wide range of differentiated cell types, including neuroectoderm, mesoderm, and trophectoderm but not endoderm. We also demonstrate that elevating the levels of Sox2 quickly downregulates several developmentally regulated genes, including Nanog, and a newly identified Sox2:Oct‐3/4 target gene, Lefty1. Together, these data argue that the self‐renewal of ES cells requires that Sox2 levels be maintained within narrow limits. Thus, Sox2 appears to function as a molecular rheostat that controls the expression of a critical set of embryonic genes, as well as the self‐renewal and differentiation of ES cells.

Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression. These small, non-coding RNAs are believed to regulate more than a third of all protein coding genes, and they have been implicated in the control of virtually all biological processes, including the biology of stem cells. The essential roles of miRNAs in the control of pluripotent stem cells were clearly established by the finding that embryonic stem (ES) cells lacking proteins required for miRNA biogenesis exhibit defects in proliferation and differentiation. Subsequently, the function of numerous miRNAs has been shown to control the fate of ES cells and to directly influence critical gene regulatory networks controlled by pluripotency factors Sox2, Oct4, and Nanog. Moreover, a growing list of tissue-specific miRNAs, which are silenced or not processed fully in ES cells, has been found to promote differentiation upon their expression and proper processing. The importance of miRNAs for ES cells is further indicated by the exciting discovery that specific miRNA mimics or miRNA inhibitors promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Although some progress has been made during the past two years in our understanding of the contribution of specific miRNAs during reprogramming, further progress is needed since it is highly likely that miRNAs play even wider roles in the generation of iPS cells than currently appreciated. This review examines recent developments related to the roles of miRNAs in the biology of pluripotent stem cells. In addition, we posit that more than a dozen additional miRNAs are excellent candidates for influencing the generation of iPS cells as well as for providing new insights into the process of reprogramming.

DNA microarray analyses of genes regulated during the differentiation of embryonic stem cells.

Embryonic stem (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. This makes ES cells an attractive model system for studying the initial developmental decisions that occur during embryogenesis and the molecular genetics and associated mechanisms underlying these decisions. Additionally, ES cells are of significant interest to those characterizing various gene functions utilizing transgenic and gene‐targeting techniques. 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. To this end, we have utilized Clontechs Atlas Mouse cDNA Expression Arrays to examine the expression of 588 known regulatory genes in D3 ES cells and their RA‐induced differentiated progeny. We report that nearly 50% of the regulatory genes are expressed in D3 and/or D3‐differentiated cells. Of these genes, the steady‐state levels of 18 are down‐regulated and 61 are up‐regulated by a factor of 2.5‐fold or greater. These changes in gene expression are highly reproducible and represent changes in the expression of a variety of molecular markers, including: transcription factors, growth factors and their receptors, cytoskeletal and extracellular matrix proteins, cell surface antigens, and intracellular signal transduction modulators and effectors. Mol. Reprod. Dev. 56:113–123, 2000.

Sox2 and Oct‐3/4: a versatile pair of master regulators that orchestrate the self‐renewal and pluripotency of embryonic stem cells

During the past 10 years, remarkable progress has been made in understanding the transcriptional mechanisms that control the biology of stem cells. Given the importance of stem cells in development, regenerative medicine, and cancer, it is no surprise that the pace of discovery continues to accelerate—paradigm‐shifting models proposed only a few years ago are quickly giving way to even more sophisticated models of regulation. This review summarizes some of the major advances made in delineating the roles of two transcription factors, Sox2 and Oct‐3/4, in stem cell biology. Additionally, unanswered questions related to their mechanisms of action are discussed. When viewed together, it is evident that Sox2 and Oct‐3/4 exhibit the major properties expected of master regulators. They are each essential for mammalian development, they help regulate the transcription of other genes that are essential for development, and they influence their own transcription by both positive and negative feedback loops. Moreover, small changes in the levels of either Sox2 or Oct‐3/4 trigger the differentiation of embryonic stem (ES) cells. Thus, each functions as a molecular rheostat to control the self‐renewal and pluripotency of ES cells. Overall, understanding how Sox2 and Oct‐3/4 function mechanistically will not only provide important insights into stem cells in general, but should also have a significant impact on our understanding of induced pluripotent stem (iPS) cells and, hence, the emerging field of regenerative medicine. Copyright

Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes

Recent studies have identified large sets of genes in embryonic stem and embryonal carcinoma 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 cooperatively to stimulate the transcription of their own genes as well as a network of genes required for embryogenesis. Moreover, small changes in the levels of Sox2:Oct-3/4 target genes alter the fate of stem cells. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, little is known about the mechanisms that prevent their overexpression. Here, we demonstrate that elevating Sox2 levels inhibits the endogenous expression of five Sox2:Oct-3/4 target genes. In addition, we show that Sox2 repression is dependent on the binding sites for Sox2 and Oct-3/4. We also demonstrate that inhibition is dependent on the C-terminus of Sox2, which contains its transactivation domain. Finally, our studies argue that overexpression of neither Oct-3/4 nor Nanog broadly inhibits Sox2:Oct-3/4 target genes. Collectively, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes and argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network.

Specific down-regulation of annexin II expression in human cells interferes with cell proliferation

The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.

Proteomic analysis of Sox2-associated proteins during early stages of mouse embryonic stem cell differentiation identifies Sox21 as a novel regulator of stem cell fate

Small increases in the levels of master regulators, such as Sox2, in embryonic stem cells (ESC) have been shown to promote their differentiation. However, the mechanism by which Sox2 controls the fate of ESC is poorly understood. In this study, we employed multidimensional protein identification technology and identified >60 nuclear proteins that associate with Sox2 early during ESC differentiation. Gene ontology analysis of Sox2‐associated proteins indicates that they participate in a wide range of 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. Moreover, we examined the impact of manipulating the expression of a Sox2‐associated protein on the fate of ESC. Using ESC engineered for inducible expression of Sox21, we show that ectopic expression of Sox21 in ESC induces their differentiation into specific cell types, including those that express markers representative of neurectoderm and heart development. Collectively, these studies provide new insights into the range of molecular processes through which Sox2 is likely to influence the fate of ESC and provide further support for the conclusion that the expression of Sox proteins in ESC must be precisely regulated. Importantly, our studies also argue that Sox2, along with other pluripotency‐associated transcription factors, is woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC. STEM CELLS 2010;28:1715–1727

Embryonal carcinoma cell growth and differentiation. Production of and response to molecules with transforming growth factor activity.

Transforming growth factors are known to induce anchorage-independent growth of non-transformed cells, and are released by a variety of cells, including MSV-transformed cells. This study demonstrates that the differentiated cells derived from F9 and PC-13 embryonal carcinoma cells, but not the parental cells themselves, respond by increased growth to several factors released by MSV-transformed cells, including partially purified sarcoma growth factor. The chemical properties of the growth-promoting activity are shown to match the chemical properties of the transforming growth factors released by MSV-transformed cells. Furthermore, F9 and PC-13 embryonal carcinoma cells, which do not respond to factors released by MSV-transformed cells, are shown to release factors with transforming growth factor activity. Based on the close relationship between mouse embryonal carcinoma cells and cells of early mouse embryos, it is suggested that molecules with transforming growth factor activity may play a role during the early stages of mammalian development.

Identification of the Transactivation Domain of the Transcription Factor Sox-2 and an Associated Co-activator

The importance of interactions between Sox and POU transcription factors in the regulation of gene expression is becoming increasingly apparent. Recently, many examples of the involvement of Sox-POU partnerships in transcription have been discovered, including a partnership between Sox-2 and Oct-3. Little is known about the mechanisms by which these factors modulate transcription. To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2. This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4. Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus. We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the transcriptional activation mediated by the combined action of Sox-2 and Oct-3. In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects. Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays. Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300.

Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells☆

In this report, we demonstrate that F9 and PC-13 embryonal carcinoma (EC) cells do not bind significant amounts of platelet-derived growth factor (PDGF), whereas the endoderm-like differentiated cells derived from EC cells do. The F9-differentiated cells exhibit approximately 8300 receptors per cell, with an apparent dissociation constant of 30 pM. Two endoderm-like cell lines, PSA-5E and PYS-2, also bind PDGF and exhibit approximately 4800 and 23,500 receptors per cell, respectively. The lack of PDGF binding by the parental EC cells is consistent with their release of a factor(s) that is closely related to PDGF. This factor(s) competes with PDGF for binding to membrane receptors and is recognized by antibodies raised against PDGF. However, this factor(s) does not appear to be antigenically identical to PDGF. We also show that production of this PDGF-like factor(s) is reduced more than 90% when F9 EC cells differentiate into cells that bind PDGF. Thus, our findings indicate that EC cells release a factor(s) that should be capable of binding to their differentiated cells. This raises the possibility that PDGF, or a closely related factor, can influence cell proliferation and/or cell behavior of early embryonic cells.