Phillip J. Wilder

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.

The SOX2-interactome in brain cancer cells identifies the requirement of MSI2 and USP9X for the growth of brain tumor cells.

Medulloblastomas and glioblastomas, the most common primary brain tumors in children and adults, respectively, are extremely difficult to treat. Efforts to identify novel proteins essential for the growth of these tumors may help to further our understanding of the biology of these tumors, as well as, identify targets for future therapies. The recent identification of multiple transcription factor-centric protein interaction landscapes in embryonic stem cells has identified numerous understudied proteins that are essential for the self-renewal of these stem cells. To identify novel proteins essential for the fate of brain tumor cells, we examined the protein interaction network of the transcription factor, SOX2, in medulloblastoma cells. For this purpose, Multidimensional Protein Identification Technology (MudPIT) identified >280 SOX2-associated proteins in the medulloblastoma cell line DAOY. To begin to understand the roles of SOX2-associated proteins in brain cancer, we focused on two SOX2-associated proteins, Musashi 2 (MSI2) and Ubiquitin Specific Protease 9x (USP9X). Recent studies have implicated MSI2, a putative RNA binding protein, and USP9X, a deubiquitinating enzyme, in several cancers, but not brain tumors. We demonstrate that knockdown of MSI2 significantly reduces the growth of DAOY cells as well as U87 and U118 glioblastoma cells. We also demonstrate that the knockdown of USP9X in DAOY, U87 and U118 brain tumor cells strongly reduces their growth. Together, our studies identify a large set of SOX2-associated proteins in DAOY medulloblastoma cells and identify two proteins, MSI2 and USP9X, that warrant further investigation to determine whether they are potential therapeutic targets for brain cancer.

NF-κB Regulates BCL3 Transcription in T Lymphocytes Through an Intronic Enhancer

Exposure to soluble protein Ags in vivo leads to abortive proliferation of responding T cells. In the absence of a danger signal, artificially provided by adjuvants, most responding cells die, and the remainder typically become anergic. The adjuvant-derived signals provided to T cells are poorly understood, but recent work has identified BCL3 as the gene, of those tested, with the greatest differential transcriptional response to adjuvant administration in vivo. As an initial step in analyzing transcriptional responses of BCL3 in T cells, we have identified candidate regulatory regions within the locus through their evolutionary conservation and by analysis of DNase hypersensitivity. An evolutionarily conserved DNase hypersensitive site (HS3) within intron 2 was found to act as a transcriptional enhancer in response to stimuli that mimic TCR activation, namely, PHA and PMA. In luciferase reporter gene constructs transiently transfected into the Jurkat T cell line, the HS3 enhancer can cooperate not only with the BCL3 promoter, but also with an exogenous promoter from herpes simplex thymidine kinase. Deletional analysis revealed that a minimal sequence of ∼81 bp is required for full enhancer activity. At the 5′ end of this minimal sequence is a κB site, as confirmed by EMSAs. Mutation of this site in the context of the full-length HS3 abolished enhancer activity. Cotransfection with NF-κB p65 expression constructs dramatically increased luciferase activity, even without stimulation. Conversely, cotransfection with the NF-κB inhibitor IκBα reduced activation. Together, these results demonstrate a critical role for NF-κB in BCL3 transcriptional up-regulation by TCR-mimetic signals.

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.

KNOCKOUT OF ONE ACETYLCHOLINESTERASE ALLELE IN THE MOUSE

One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.

Crystal Structure of Mouse Elf3 C-terminal DNA-binding Domain in Complex with Type II TGF-β Receptor Promoter DNA

The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-beta receptor gene (TbetaR-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, the A-site and the B-site. Here, we report the 2.2 A resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-beta receptor promoter DNA (mTbetaR-II(DNA)). Elf3 contacts the core GGAA motif of the B-site from a major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity.

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.

Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications

Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating thein vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.