Structural basis of Ets1 activation by Runx1.

Runx1 is required for definitive hematopoiesis and is well known for its frequent chromosomal translocations and point mutations in leukemia. Runx1 regulates a variety of genes via Ets1 activation on an Ets1•Runx1 composite DNA sequence. The structural basis of such regulation remains unresolved. To address this problem, we determined the crystal structure of the ternary complex containing Runx1(1-242) and Ets1(296-441) bound to T-cell receptor alpha (TCRα) enhancer DNA. In the crystal, an Ets1-interacting domain of Runx1 is bound to the Ets1 DNA-binding domain and displaced an entire autoinhibitory module of Ets1, revealing a novel mechanism of Ets1 activation. The DNA-binding and transcriptional studies with a variety of structure-guided Runx1 mutants confirmed a critical role of direct Ets1•Runx1 interaction in Ets1 activation. More importantly, the discovered mechanism provides a plausible explanation for how the Ets1•Runx1 interaction effectively activates not only a wild-type Ets1, but also a highly inhibited phosphorylated form of Ets1.

Isolation and characterization of the murine transforming growth factor-beta2 promoter.

This report describes the isolation and characterization of the 5' flanking region of the murine transforming growth factor beta-2 (TGF-beta2) 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-beta2 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-beta2 promoter/reporter constructs was generated to identify regulatory regions of the gene. As in the case of the human TGF-beta2 gene, sequences just upstream of the TATA box, including the CRE/ATF site, actively stimulate the murine TGF-beta2 promoter. However, unlike the human TGF-beta2 gene, the 5' flanking region of the murine TGF-beta2 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.

Transcriptional regulation of the transforming growth factor-beta2 gene in glioblastoma cells.

The expression of transforming growth factor-beta2 (TGF-beta2) appears to play a strong role in the establishment and progression of glial tumors. In particular, elevated expression of TGF-beta2 appears to be responsible for the impaired cell-mediated immunity often observed in patients with a glioblastoma. This study examined the regulation of the TGF-beta2 at the transcriptional level in the U87MG glioblastoma cell line. We demonstrate that a cAMP response element/activating transcription factor (CRE/ATF) site and an E-box motif located just upstream of the transcription start site are essential for the transcription of the TGF-beta2 gene in U87MG cells. Gel mobility analysis determined that activating transcription factor-1, and possibly cAMP-responsive element binding protein, binds to the CRE/ATF site, and upsteam stimulatory factor (USF) 1 and USF2 bind to the E-box motif. Interestingly, expression of a dominant negative USF protein down-regulates TGF-beta2 activity by 80-95% in glioblastoma cells. We conclude that the binding of transcription factors, in particular the USF proteins, to the TGF-beta2 promoter is essential for its expression and possibly its up-regulation in glioblastomas.

Effects of three Sp1 motifs on the transcription of the FGF-4 gene.

Previous studies have shown that the transcription of the fibroblast growth factor-4 (FGF-4) gene is regulated by a powerful enhancer located approximately three kilobases downstream of the transcription start site. Several conserved cis-regulatory elements in the promoter and the enhancer have been identified, including two Sp1 motifs located in the promoter and one Sp1 motif located in the enhancer. Each of these Sp1 motifs has been shown previously to bind the transcription factors Sp1 and Sp3 in vitro. The main objective of this study was to examine the potential interaction of the FGF-4 promoter and enhancer Sp1 motifs. Using site-directed mutagenesis, we demonstrate that disruption of these sites, individually or in combination, reduce the expression of FGF-4 promoter/reporter gene constructs in embryonal carcinoma cells. Importantly, we demonstrate that disruption of the enhancer Sp1 motif exerts a more pronounced effect on the expression of these constructs than disruption of the promoter Sp1 motifs. We also demonstrate that changing the spacing and the stereo-alignment of the enhancer Sp1 motif, relative to the other cis-regulatory elements of the enhancer, has little effect on the ability of the enhancer to stimulate transcription. Furthermore, embryonic stem cells that contain two disrupted Sp1 alleles were used to demonstrate that the transcription factor Sp1 is not necessary for expression of the endogenous FGF-4 gene. Finally, the significance of these findings relative to a looping model for the transcriptional activation of the FGF-4 gene is discussed.

Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase.

Acetylcholinesterase (AChE; EC 3.1.1.7) is the primary terminator of nerve impulse transmission at cholinergic synapses and is believed to play an important role in neural development. Targeted deletion of four exons of the ACHE gene reduced AChE activity by half in heterozygous mutant mice and totally eliminated AChE activity in nullizygous animals. Butyrylcholinesterase (EC 3.1.1.8) activity was normal in AChE -/- mice. Although nullizygous mice were born alive and lived up to 21 days, physical development was delayed. The neuromuscular junction of 12-day-old nullizygous animals appeared normal in structure. Nullizygous mice were highly sensitive to the toxic effects of the organophosphate diisopropylfluorophosphate and to the butyrylcholinesterase-specific inhibitor bambuterol. These findings indicate that butyrylcholinesterase and possibly other enzymes are capable of compensating for some functions of AChE and that the inhibition of targets other than AChE by organophosphorus agents results in death.

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 Clontech's 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.

Isolation, characterization, and differential expression of the murine Sox-2 promoter.

Sox proteins are expressed at many stages of development and in numerous tissues. The transcription factor Sox-2 is first expressed throughout the inner cell mass and subsequently becomes localized to the primitive ectoderm, developing central nervous system, and the lens. Sox-2 is also highly expressed in F9 embryonal carcinoma cells, but becomes undetectable following differentiation of these cells. In this study, we have isolated, sequenced, and performed the first characterization of the Sox-2 promoter of any species. Approximately 2kb of the Sox-2 5'-flanking region has been sequenced and the primary transcription start site mapped by primer extension analysis. Additionally, two positive regulatory regions within the promoter region have been identified. We also show that expression of Sox-2 promoter/reporter gene constructs is reduced in differentiated EC cells as compared to their undifferentiated counterparts. Furthermore, we have identified a consensus inverted CCAAT box motif present in the Sox-2 promoter. Mutagenesis of this site significantly reduces the expression of Sox-2 promoter/reporter constructs. We also demonstrate that this CCAAT box motif can bind the trimeric transcription factor NF-Y.

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.

Transcriptional regulation of the transforming growth factor-beta2 promoter by cAMP-responsive element-binding protein (CREB) and activating transcription factor-1 (ATF-1) is modulated by protein kinases and the coactivators p300 and CREB-binding protein.

Transcription of the transforming growth factor-beta2 (TGF-beta2) gene is dependent on a cAMP-response element/activating transcription factor (CRE/ATF) site that is bound by CREB and ATF-1 as well as an E-box motif that is bound by upstream stimulatory factors 1 and 2 (USF1 and USF2). To identify additional factors involved in the expression of the TGF-beta2 gene, we employed F9 embryonal carcinoma (EC) cells, which express TGF-beta2 only after the cells differentiate. We show that overexpression of the transcription factors, CREB, ATF-1, USF1, and USF2 dramatically increases TGF-beta2 promoter activity in F9-differentiated cells. We further show that the coactivators p300 and CBP up-regulate the TGF-beta2 promoter when CREB and ATF-1 are expressed in conjunction with protein kinases that phosphorylate CREB on serine 133 and ATF-1 on serine 63. Importantly, we identify the presence of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. This phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells and is likely to play an important role in the activation of the TGF-beta2 gene.

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.

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.

Growth regulatory factors and carcinogenesis: the roles played by transforming growth factor beta, its receptors and signaling pathways.

It is widely recognized that growth factors play critical roles in cell proliferation and differentiation. In the early 1980s, several members of the transforming growth factor-beta (TGF-beta) superfamily were identified and subsequently shown to play important roles in many diseases, in particular cancer. Efforts to understand how TGF-beta exerts its effects led to identification of TGF-beta-receptors and several downstream signaling pathways activated by this family of growth factors. This review provides an overview of TGF-beta-receptors and its downstream signaling pathways. As part of this discussion, this review indicates that inactivation of TGF-beta-receptors or components of their signaling pathways is often a target during carcinogenesis and that mutations or altered expression at any step along this complex, growth regulatory pathway can lead to aberrant cell proliferation. Lastly, the Cancer Genome Anatomy Project is briefly discussed, in particular how it may help to identify aberrant growth factor expression during carcinogenesis and improve the diagnosis of cancer patients.

Effects of differentiation on the transcriptional regulation of the FGF-4 gene: critical roles played by a distal enhancer.

Embryonal carcinoma (EC) cells are used widely as a model system for studying the expression of developmentally regulated genes, in particular genes that are regulated at the transcriptional level when EC cells differentiate. This review focuses on the molecular mechanisms that govern the transcription of the fibroblast growth factor-4 (FGF-4) gene, which appears to be the first FGF expressed during mammalian development. Interest in this gene has increased considerably with the finding that FGF-4 is essential for mammalian embryogenesis. The FGF-4 gene has also generated considerable interest because it is inhibited at the transcriptional level when EC cells undergo differentiation and because this gene is regulated by a powerful distal enhancer located 3 kb downstream of the transcription start site in the last exon of the gene. Hence, study of the FGF-4 gene is likely to shed light on the molecular mechanisms by which distal enhancers regulate gene expression. In addition to being regulated by the downstream enhancer, the expression of this gene is influenced by a regulatory region located just upstream of the transcription start site, which contains two Sp1 motifs and a CCAAT box motif. Examination of the downstream enhancer has identified three functional cis-regulatory elements: a high mobility group (HMG) protein binding motif, an octamer binding motif, and an Sp1 motif, which are likely to bind Sox-2, Oct-3, and Sp1/Sp3, respectively, in vivo. Interestingly, Sox-2 and Oct-3 expression, like FGF-4 expression, decreases when EC cells differentiate, which suggests that the loss of these transcription factors is responsible, at least in part, for the transcriptional turn-off of the FGF-4 gene. In view of these and other findings, we present a model for the differential expression of the FGF-4 gene that includes not only the contributions of specific transcription factors, but also the contribution of chromatin structure before and after differentiation.

Transcriptional activation of the type II transforming growth factor-beta receptor gene upon differentiation of embryonal carcinoma cells.

Previously, it has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type-beta receptors. Here, we show that the type II receptor (TbetaR-II) gene is activated at the transcriptional level when EC cells differentiate. We show that the differentiated cells, but not the parental EC cells, express transcripts for TbetaR-II. In addition, the expression of TbetaR-II promoter/reporter gene constructs are elevated dramatically when EC cells differentiate and we identify at least two positive and two negative regulatory regions in the 5' flanking region of the TbetaR-II gene. Moreover, we identify a cAMP response element/activating transcription factor site that acts as a positive cis-regulatory element in the TbetaR-II promoter, and we demonstrate that the transcription factor ATF-1 binds to this site and strongly stimulates the expression of the TbetaR-II promoter/reporter gene constructs when ATF-1 is overexpressed in EC-derived differentiated cells. Equally important, we identify a negative regulatory element in a 53-base pair region that had previously been shown to inhibit strongly the expression of TbetaR-II promoter/reporter gene constructs. Specifically, we demonstrate that this region, which contains an inverted CCAAT box motif, binds the transcription factor complex NF-Y (also referred to as CBF) in vitro. Furthermore, expression of a dominant-negative NF-YA mutant protein, which prevents DNA binding by NF-Y, enhances TbetaR-II promoter expression. Together, these studies suggest that the transcription factors ATF-1 and NF-Y play important roles in the regulation of the TbetaR-II gene.

Role of the transcription factor Sox-2 in the expression of the FGF-4 gene in embryonal carcinoma cells.

It has been shown previously that the FGF-4 gene is regulated by a powerful downstream enhancer in embryonal carcinoma (EC) cells. This enhancer contains an essential HMG motif; however, the transcription factor that binds to the HMG motif in EC cells has not been determined definitively. In earlier studies, this HMG motif was shown to bind a heat-stable, redox-insensitive factor expressed by F9 EC cells. Others have proposed that the transcription factor Sox-2 binds to the FGF-4 enhancer HMG motif. In this study, we demonstrate that the N-terminal half of Sox-2, which contains the DNA binding domain, binds to the FGF-4 enhancer HMG motif and we show that this binding is unaffected by heat and oxidation. In addition, we employed two experimental approaches to demonstrate that Sox-2 regulates the transcription of the FGF-4 gene in EC cells. As part of these studies, an expression plasmid that codes for a dominant-negative form of Sox-2 was used in transient expression assays. In other experiments, a Sox-2 antisense expression plasmid was used. When co-transfected into F9 EC cells along with an FGF-4 promoter/reporter gene construct, each expression plasmid caused a significant reduction in reporter activity. Our studies also demonstrate that Sox-2 affects the expression of the FGF-4 gene in the multipotent EC cell line, P19. Taken together, these studies argue strongly that Sox-2 plays an important role in the expression of the FGF-4 gene in vivo.

NF-Y binds to the CCAAT box motif of the FGF-4 gene and promotes FGF-4 expression in embryonal carcinoma cells.

FGF-4 appears to be the first fibroblast growth factor (FGF) expressed during embryogenesis, and its expression is critical for early mammalian development. FGF-4 is expressed in the embryonic cell lines, F9, D3, and NT2/D1; but its expression in these cells is repressed upon differentiation. Transcription of the FGF-4 gene in embryonic cells is regulated by an enhancer in the third exon and by a positive regulatory region upstream of the transcription start site. A CCAAT box motif within the positive regulatory region has been shown to support FGF-4 expression, but the factor that binds to this site in vivo has not been identified. In this report, we demonstrate that the transcription factor complex NF-Y binds to the FGF-4 CCAAT box motif when nuclear extracts from each of the embryonic cell lines and their differentiated cells were examined by gel mobility shift analyses. Importantly, we demonstrate that expression of a dominant-negative NF-YA mutant protein reduces the expression of FGF-4 promoter/reporter gene constructs in F9 EC cells. Hence, we provide strong evidence that the transcription factor NF-Y is involved in the expression of the FGF-4 gene.

Transcriptional regulation of the TGF-beta 2 gene in choriocarcinoma cells and breast carcinoma cells: differential utilization of Cis-regulatory elements.

Previous studies have shown that the transcription of the TGF-beta 2 gene is controlled by at least one negative and two positive regulatory regions in differentiated cells derived from both embryonal carcinoma cells and embryonic stem cells. The use of TGF-beta 2 promoter/reporter gene constructs has also identified a CRE/ATF motif near the TATA box that appears to heavily influence the transcription of the TGF-beta 2 gene. In this study, two choriocarcinoma cell lines, JAR and JEG-3, and the breast cancer cell line, MCF-7, were used to determine whether differences exist in the transcriptional regulation of the TGF-beta 2 gene. We demonstrated that both similarities and differences exist in the transcriptional regulation of this gene. Common to all cells examined to date, the positive regulatory region just upstream of the TATA box contains an essential CRE/ATF motif that binds at least one transcription factor, ATF-1, in gel mobility shift assays. However, we did not detect ATF-2 binding to this site with any of the nuclear extracts used. We also determined that the effect of the region between -187 and -78 (relative to the transcription start site) is cell type dependent. Previous studies have shown that this region acts to reduce the activity of the TGF-beta 2 promoter in differentiated cells derived from embryonal carcinoma cells and embryonic stem cells. In direct contrast, this region acts as a strong positive regulatory region in JAR, JEC-3, and MCF-7 cells. The mechanisms responsible for these differing effects remain to be established. Interestingly, this region does not appear to contain sequence motifs that bind known transcription factors. Thus, this region is likely to bind one or more novel transcription factors or contain novel recognition sites for known transcription factors.

DNA sequence and nucleosome placement on the murine fibroblast growth factor-4 gene.

A large portion of the 5' flanking region of the fibroblast growth factor-4 (FGF-4) gene has been sequenced, but only 38 bp of sequence information past the end of the last exon of this gene has been described. In this study, a total of 2769 bp of nucleotide sequence down-stream of the last exon of FGF-4 (GenBank #U43515) was determined by a combination of deletional subcloning and primer walking. In addition, we have used the theoretical algorithm of Calladine and Drew to predict the rotational positioning of nucleosomes throughout the entire FGF-4 gene.

Inactivation of the FGF-4 gene in embryonic stem cells alters the growth and/or the survival of their early differentiated progeny.

Previous studies have shown that early mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, we prepared genetically engineered embryonic stem (ES) cells, which are unable to produce FGF-4. Specifically, we describe the isolation and characterization of ES cells with both FGF-4 alleles inactivated. The FGF-4-/- ES cells do not require FGF-4 to proliferate in vitro, and addition of FGF-4 to the medium has little or no effect on their growth. Thus, FGF-4 does not appear to act as an autocrine growth factor for cultured ES cells. We also demonstrate that FGF-4-/- ES cells, like their unmodified counterparts, are capable of forming highly complex tumors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, we demonstrate that the FGF-4-/- ES cells can differentiate in vitro after exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importantly, addition of FGF-4 to the culture medium dramatically increases the number of differentiated cells derived from the FGF-4-/- ES cells, in particular cells with many of the properties of parietal extraembryonic endoderm. Finally, we demonstrate that there are differences in the RNA profiles expressed by the differentiated progeny formed in vitro from FGF-4-/- ES cells and FGF-4+/+ ES cells when they are cultured with FGF-4. Taken together, the studies described in this report indicate that certain lineages formed in vitro are affected by the inactivation of the FGF-4 gene, in particular specific cells that form during the initial stage of ES cell differentiation. Thus, ES cells with both FGF-4 alleles inactivated should shed light on the important roles of FGF-4 during the early stages of mammalian development and help determine why FGF-4-/- embryos die shortly after implantation.

Transcription of the transforming growth factor-beta2 gene is dependent on an E-box located between an essential cAMP response element/activating transcription factor motif and the TATA box of the gene.

Transforming growth factor-beta2 (TGF-beta2) is an important regulator of cell proliferation and differentiation; however, its transcriptional regulation is not well understood. Here we report characterization of an essential E-box motif, positioned at -50/-45 between a previously described functional cAMP response element/activating transcription factor site and the TATA box of the human TGF-beta2 promoter. By site-directed mutagenesis, we demonstrate that this E-box motif is necessary for the promoter activity, not only in differentiated cells derived from embryonal carcinoma cells, but also in choriocarcinoma cells and in MCF-7 breast carcinoma cells. We also demonstrate that the transcription factors USF1 and USF2 bind to this E-box motif in vitro when nuclear extracts from each of these cell lines are examined by gel retardation assays. Moreover, using a dominant-negative USF2 protein, we show that USF proteins are critical for TGF-beta2 promoter activity in vivo. The importance of the E-box motif described in this study is supported by the presence of an E-box motif in the same position in the chicken TGF-beta2 gene promoter.