Michelle Desler

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.

ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study, we demonstrate that Y‐27632, a specific inhibitor of Rho kinase (ROCK) activity, significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore, treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably, hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y‐27632. Additionally, we determined that Y‐27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus, Y‐27632 provides a means to “kick‐start” slow‐growing human pluripotent stem cells, especially after being thawed from frozen stocks. Together, these results argue that Y‐27632 is a useful tool in overcoming obstacles to studies involving the cultivation of both hES cells and human iPS cells. Mol. Reprod. Dev. 76: 722–732, 2009.

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.

Elevating SOX2 levels deleteriously affects the growth of medulloblastoma and glioblastoma cells.

Medulloblastomas and glioblastomas are devastating tumors that respond poorly to treatment. These tumors have been shown to express SOX2 and overexpression of SOX2 has been correlated with poor prognosis. Although knockdown of SOX2 impairs the growth and tumorigenicity of brain tumor cells, it was unclear how elevating SOX2 levels would affect their fate. Interestingly, studies conducted with neural stem cells have shown that small increases or decreases in the level of this transcription factor significantly alter their fate. Here, we report that elevating SOX2 3-fold above endogenous levels in U87 and U118 glioblastoma, and DAOY medulloblastoma cells significantly impairs their ability to proliferate. We extended these findings and determined that elevating SOX2 in DAOY cells remodels their cell-cycle profile by increasing the proportion of cells in the G1-compartment, and induces the expression of genes associated with differentiation. Furthermore, we show that elevating SOX2 leads to a dramatic induction of CD133 expression in DAOY cells, yet inhibits the ability of both CD133+ and CD133− cells to form neurospheres. Together, these findings argue that SOX2 levels must be carefully controlled in glioblastomas and medulloblastomas to maintain their fate. Equally important, our data suggests that increases in the expression of SOX2 during brain tumor progression are likely to be linked closely with changes in other critical genes that work in concert with SOX2 to enhance the tumorigenicity of brain tumors. Importantly, we demonstrate that this is also likely to be true for other cancers that express SOX2. Moreover, these studies demonstrate the advantage of using inducible promoters to study the effects of SOX2 elevation, as compared to gene expression systems that rely on constitutive expression.

Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA

Ets1 is a member of the Ets family of transcription factors.

Different Domains of the Transcription Factor ELF3 Are Required in a Promoter-specific Manner and Multiple Domains Control Its Binding to DNA

Elf3 is an epithelially restricted member of the ETS transcription factor family, which is involved in a wide range of normal cellular processes. Elf3 is also aberrantly expressed in several cancers, including breast cancer. 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 the transactivation domain that behaves as an autoinhibitory domain. Interestingly, disruption of the transactivation domain relieves the autoinhibition of Elf3 and enhances Elf3 binding to DNA. On the basis of these studies, we suggest a model for autoinhibition of Elf3 involving intramolecular interactions. Importantly, this model is consistent with our finding that the N-terminal region of Elf3, which contains the transactivation domain, interacts with its C terminus, which contains the ETS domain. In parallel studies, we demonstrate that residues flanking the N- and C-terminal sides of the ETS domain of Elf3 are crucial for its binding to DNA. Our studies also show that an AT-hook domain, as well as the serine- and aspartic acid-rich domain but not the pointed domain, is necessary for Elf3 activation of promoter activity. Unexpectedly, we determined that one of the AT-hook domains is required in a promoter-specific manner.

Differential activity of the FGF-4 enhancer in F9 and P19 embryonal carcinoma cells

Transcription factors Oct‐3/4 and Sox2 behave as global regulators during mammalian embryogenesis. They work together by binding co‐operatively to closely spaced HMG and POU motifs (HMG/POU cassettes). Recently, it was suggested that a critical Sox2:Oct‐3/4 target gene, FGF‐4, is expressed at lower levels in P19 than in F9 embryonal carcinoma (EC) cells, due to lower levels of Sox2 in P19 than in F9 cells. We tested this possibility to better understand how FGF‐4 expression is modulated during development. Although we found that P19 EC cells express ∼10‐fold less FGF‐4 mRNA than F9 EC cells, we determined that Sox2 levels do not differ markedly in F9 and P19 EC cells. We also determined that Sox2 and Oct‐3/4 work together equally well in both EC cell lines. Moreover, in contrast to an earlier prediction based on in vitro binding studies, we demonstrate that the function of the HMG/POU cassettes of the FGF‐4 and UTF1 genes does not differ significantly in these EC cell lines when tested in the context of a natural enhancer. Importantly, we determined that the FGF‐4 promoter is highly responsive to a heterologous enhancer in both EC cell lines; whereas, the FGF‐4 enhancer is 7‐ to 10‐fold less active in P19 than in F9 EC cells. Because F9 and P19 EC cells are likely to represent cells at different stages of mammalian development, we suggest that this difference in FGF‐4 enhancer activity may reflect a mechanism used to decrease, but not abolish, FGF‐4 expression as the early embryo develops. J. Cell. Physiol.

Differential regulation of the Oct‐3/4 gene in cell culture model systems that parallel different stages of mammalian development

Oct‐3/4 is an essential transcription factor that regulates stem cell fate during embryogenesis. Previous reports have shown that the Oct‐3/4 gene utilizes different enhancers to regulate its expression as development proceeds. However, the cis‐elements contributing to the differential activity of these enhancers require further study. Here, we investigated the function of the HMG/POU cassette and LRH‐1 site present in the distal enhancer (DE) and the proximal enhancer, respectively. F9 and P19 EC cells were the focus of this study because their differential utilization of Oct‐3/4 enhancers parallels the use of these enhancers during different stages of development. We determined that the LRH‐1 site functions as a positive and a negative cis‐regulatory element in P19 and F9 EC cells, respectively. Furthermore, we determined that the HMG/POU cassette in the DE strongly activates the Oct‐3/4 promoter in F9 cells, but is a much weaker positive regulatory element in P19 cells. Given that HMG/POU cassettes play key roles in the regulation of at least seven essential genes, the Oct‐3/4 HMG/POU cassette was examined more closely by focusing on Sox2, which can bind to HMG/POU cassettes. Although chromatin immunoprecipitation demonstrated that Sox2 binds to the Oct‐3/4 gene equally well in both EC cell lines, tethering Sox2 to the region of the HMG/POU cassette only activated the Oct‐3/4 promoter in F9 EC cells. These and other findings suggest that the differential activity of the HMG/POU cassette of the Oct‐3/4 gene in EC cells is due to differential action of Sox2 and its associated co‐factors. Mol. Reprod. Dev. 75: 1247–1257, 2008.

Regulation of the Nanog Gene by Both Positive and Negative cis-Regulatory Elements in Embryonal Carcinoma Cells and Embryonic Stem Cells

The transcription factor Nanog is essential for mammalian embryogenesis, as well as the pluripotency of embryonic stem (ES) cells. Work with ES cells and embryonal carcinoma (EC) cells previously identified positive and negative cis‐regulatory elements that influence the activity of the Nanog promoter, including adjacent cis‐regulatory elements that bind Sox2 and Oct‐3/4. Given the importance of Nanog during mammalian development, we examined the cis‐regulatory elements required for Nanog promoter activity more closely. In this study, we demonstrate that two positive cis‐regulatory elements previously shown to be active in F9 EC cells are also active in ES cells. We also identify a novel negative regulatory region that is located in close proximity to two other positive Nanog cis‐regulatory elements. Although this negative regulatory region is active in F9 EC cells and ES cells, it is inactive in P19 EC cells. Furthermore, we demonstrate that one of the positive cis‐regulatory elements active in F9 EC cells and ES cells is inactive in P19 EC cells. Together, these and other studies suggest that Nanog transcription is regulated by the interplay of positive and negative cis‐regulatory elements. Given that P19 appears to be more closely related to a later developmental stage of mammalian development than F9 and ES cells, differential utilization of cis‐regulatory elements may reflect mechanisms used during development to achieve the correct level of Nanog expression as embryogenesis unfolds. Mol. Reprod. Dev. 76: 173–182, 2009.

Isolation and characterization of the murine transforming growth factor-β2 promoter

Abstract This report describes the isolation and characterization of the 5′ flanking region of the murine transforming growth factor beta-2 ( TGF- β 2 ) gene. A genomic clone containing the promoter region of the gene was isolated after screening a bacteriophage P1 genomic library. The resulting clone was sequenced and compared to promoters for the human and chicken TGF- β 2 genes. The sequence located near the transcription start site is highly conserved. It includes a TATA box, an E-box, and a largely conserved CRE/ATF site. A series of murine TGF- β 2 promoter/reporter constructs was generated to identify regulatory regions of the gene. As in the case of the human TGF- β 2 gene, sequences just upstream of the TATA box, including the CRE/ATF site, actively stimulate the murine TGF- β 2 promoter. However, unlike the human TGF- β 2 gene, the 5′ flanking region of the murine TGF- β 2 gene contains a long alternating purine/pyrimidine repeat that unexpectedly exerts a strong positive effect on its promoter. This is of particular interest since alternating purine/pyrimidine repeats in other promoters have been observed to be inhibitory.