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Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully re-acquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally non-functional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a sub-classification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

TEA domain (TEAD) family transcription factors are key regulators in development, tissue homeostasis and cancer progression. TEAD4 acts as a critical downstream effector of the evolutionarily conserved Hippo signaling pathway. The well-studied oncogenic protein YAP forms a complex with TEAD4 to regulate gene transcription; so does the tumor suppressor VGLL4. Although it is known that TEAD proteins can bind promoter regions of target genes through the TEA domain, the specific and detailed mechanism of DNA recognition by the TEA domain remains partially understood. Here, we report the crystal structure of TEAD4 TEA domain in complex with a muscle-CAT DNA element. The structure revealed extensive interactions between the TEA domain and the DNA duplex involving both the major and minor grooves of DNA helix. The DNA recognition helix, α3 helix, determines the specificity of the TEA domain binding to DNA sequence. Structure-guided biochemical analysis identified two major binding sites on the interface of the TEA domain-DNA complex. Mutation of TEAD4 at either site substantially decreases its occupancy on the promoter region of target genes, and largely impaired YAP-induced TEAD4 transactivation and target gene transcription, leading to inhibition of growth and colony formation of gastric cancer cell HGC-27. Collectively, our work provides a structural basis for understanding the regulatory mechanism of TEAD-mediated gene transcription.

Developing therapeutics to effectively inhibit the MYC oncoprotein would mark a key advance towards cancer patient care as MYC is deregulated in over 50% of human cancers. MYC deregulation is correlated with aggressive disease and poor patient outcome. Despite strong evidence in mouse models that inhibiting MYC would significantly impact tumour cell growth and patient survival, traditional approaches have not yet yielded the urgently needed therapeutic agents that directly target MYC. MYC functions through its interaction with MAX to regulate gene transcription by binding to E-box DNA response elements of MYC target genes. Here we used a structure-based strategy to design ME47, a small minimalist hybrid protein (MHP) able to disrupt the MAX:E-box interaction/binding and block transcriptional MYC activity. We show that inducing ME47 expression in established tumour xenografts inhibits tumour growth and decreases cellular proliferation. Mechanistically, we show by chromatin immunoprecipitation that ME47 binds to E-box binding sites of MYC target genes. Moreover, ME47 occupancy decreases MYC:DNA interaction at its cognate E-box binding sites. Taken together, ME47 is a prototypic MHP inhibitor that antagonizes tumour cell growth in vitro and in vivo and inhibits the interaction of MYC with DNA E-box elements. These results support ME47's role as a MYC inhibitor and suggest that MHPs provide an alternative therapeutic targeting system that can be used to target transcription factors important in human diseases, including cancer.

Oncogenic c-Myc has been described to modulate the expression of a subset of microRNAs (miRNAs), which include miR-22; however, the mechanism through which a miRNA controls c-Myc activity remains unclear. Here we report a novel anti-c-Myc function mediated by miR-22. Ectopically expressed miR-22 inhibited cell proliferation and anchorage-independent growth of human cancer cell lines. Microarray screening and western analyses revealed that miR-22 repressed the c-Myc-binding protein MYCBP, a positive regulator of c-Myc. Consistent with this, reporter assays showed that miR-22-mediated MYCBP gene suppression largely depends on the conserved miR-22 target site within the MYCBP 3'-untranslational region (3'UTR), implying that MYCBP mRNA is a direct miR-22 target. Depletion of MYCBP using small interfering RNA (siRNA) recapitulated the miR-22-induced anti-growth effect on tumor cells, whereas ectopically expressed MYCBP rescued cells from the growth suppression mediated by miR-22. Moreover, repression of MYCBP by miR-22 downregulated a panel of E-box-containing c-Myc target genes. Our results suggest that miR-22 acts as a tumor suppressor through direct repression of MYCBP expression and subsequent reduction of oncogenic c-Myc activities. As c-Myc inhibits the expression of miR-22, we propose a novel positive feedback loop formed by oncogenic c-Myc to accelerate cell proliferation by suppressing miR-22, a potent inhibitor of MYCBP.

Nuclear factor-kappaB (NF-kappaB) is a dynamic transcription factor that regulates important biological processes involved in cancer initiation and progression. Identifying regulators that control the half-life of NF-kappaB is important to understanding molecular processes that control the duration of transcriptional responses. In this study we identify copine-I, a calcium phospholipid-binding protein, as a novel repressor that physically interacts with p65 to inhibit NF-kappaB transcription. Knockdown of copine-I by siRNA increases tumor necrosis factor alpha-stimulated NF-kappaB transcription, while copine-I expression blocks endogenous transcription. Copine-I abolishes NF-kappaB transcription by inducing endoprotease processing of the N-terminus of p65, a process antagonized by IkappaB alpha. Copine-I stimulates endoproteolysis of p65 within a conserved region that is required for base-specific contact with DNA. p65 proteins lacking the N-terminus fail to bind to DNA and act as dominant-negative molecules that inhibit NF-kappaB transcription. Our work provides evidence that copine-I regulates the half-life of NF-kappaB transcriptional responses through a novel mechanism that involves endoproteolysis of the p65 protein.

Upregulation of human telomerase reverse transcriptase (hTERT) transcription accounts for the immortalization of greater than 85% of all human tumor cells. However, the mechanism whereby hTERT expression is activated remains unresolved. Specifically, recent data challenging the role of Myc/Max in E-box-dependent activation of hTERT expression suggests that other E-box-binding proteins regulate hTERT transcription. Indeed, we now demonstrate that two such proteins, upstream stimulatory factor (USF) 1 and 2, readily associate with two E-boxes in the hTERT promoter in vitro and in vivo primarily as heterodimers, whereas Myc/Max does not. The avid binding of USF1/2 heterodimers to these E-boxes occurs in both hTERT-positive and -negative cells. In contrast, USF1/2 activates the hTERT promoter exclusively in hTERT-positive cells in a manner that is enhanced by the coactivator p300 and attenuated upon inhibiting p38-MAP kinase, a known modulator of USF activity. Collectively, our data indicate that USF binding to the hTERT promoter may be transcriptionally neutral, or even repressive, in nonimmortalized hTERT-negative somatic cells, but stimulatory in hTERT-positive cells where USF1/2 contributes to the acquisition and maintenance of immortality.

The t(12;22) creates an MN1-TEL fusion gene leading to acute myeloid leukemia. The fusion partner TEL (ETV6) is a member of the ETS family of transcription factors. The nature of the other fusion partner, MN1, has not been investigated in detail until now. We recently described that MN1 activates the transcription activity of the moloney sarcoma virus long terminal repeat, indicating that this protein itself may act as a transcription factor. We show here that MN1 comprises multiple transcription activating domains. A search for a bound DNA sequence revealed that MN1 has affinity for retinoic acid responsive elements. A DR5 retinoic acid responsive element was observed in the LTR. The combination of MN1 and ligand-activated retinoic acid receptor leads to a synergistic induction of expression directed by the LTR. Cotransfection of MN1 with RAC3 or p300, known coactivators of retinoic acid receptors, leads to a further synergistic induction of transcription. In addition, the effect of MN1 can be inhibited by the wild-type adenovirus ElA protein that inhibits p300 function, but not by an E1A mutant lacking the p300-binding site. GAL4-MN1-mediated transcription can be enhanced directly by RAC3 and p300. Taken together, our results indicate that MN1 is a transcription coactivator rather than a sequence-specific transcription factor, and that it may stimulate RAR/RXR-mediated transcription through interaction with p160 and p300.

Inactivation of the E-cadherin cell adhesion molecule is believed critical in the development and behavior of many epithelial cancers, though mutations in the E-cadherin gene account for inactivation in only a fraction of cases. In many breast cancer lines, E-cadherin transcription is extinguished, but the role and significance of alterations in trans-acting transcription factors, promoter hypermethylation, and chromatin changes remain unresolved. To gain further insights into mechanisms underlying E-cadherin inactivation in breast cancer, we analysed somatic cell hybrids resulting from pairwise fusions between breast cancer lines with intact E-cadherin transcription (E-cad+) and lines lacking E-cadherin transcription (E-cad-). All hybrid lines failed to express E-cadherin transcripts and protein, despite the fact that E-cadherin alleles from E-cad+ lines were present in the hybrids. Elements in the proximal 108 bp of the E-cadherin promoter, when present in reporter gene constructs, were sufficient to direct strong transcription in E-cad+ breast lines, but displayed weak activity in E-cad- parental lines and hybrids. E-cadherin expression could not be restored in E-cad- lines or hybrids by treatment with a DNA demethylating agent and/or a histone deacetylase inhibitor. Our findings suggest loss of E-cadherin expression in some breast cancers may be due to dominant repression of the trans-acting pathways that regulate E-cadherin transcription.

Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent transcriptional regulator which can activate or repress specific cellular genes and has been proposed to contribute to leukemogenic processes in adult T-cell leukemia. The molecular mechanism of Tax-mediated trans-activation has been well investigated. However, trans-repression by Tax remains to be studied in detail, although it is known to require a specific DNA element such as E-box or p53 binding site. Examining possible mechanisms of trans-repression, we found that co-expression of E47 and p300 activated E-box dependent transcription and this activation was efficiently repressed by Tax. In this system, Tax bound to p300 and decreased the level of p300 complexed on the E-box element. Similarly, Tax inhibited transcription directed by p53 and CBP, reducing the level of CBP on the p53 binding site. These results indicate that Tax interferes with recruitment of CBP/p300 into protein complexes on E-box and p53 binding site through its binding to CBP/p300. In contrast to these findings, we observed that Tax increased the level of CBP on the viral 21-bp enhancer which is trans-activated by Tax. From these observations, we propose a universal mechanism for Tax-mediated trans-repression and trans-activation of transcription in which Tax binds to CBP/p300 and determines the accessibility of CBP/p300 to protein complexes on specific DNA element.

We have previously demonstrated that the core promoter of rat ribosomal RNA gene (rDNA) contains an E-box-like sequence to which the core promoter binding factor CPBF binds and that the 44 kDa subunit of this protein is immunologically related to USF1, the helix – loop – helix-zipper DNA binding protein. Further, we showed that RNA polymerase I (pol I) transcription in vitro is competed by oligonucleotides containing USF-binding site, which suggested a key role for USF in rDNA transcription. To prove the potential role of USF in pol I transcription in vivo, USF1 and USF2 homodimers and USF1/USF2 heterodimer were overexpressed in CHO cells by transfection of the respective cDNAs. Co-transfection of a plasmid containing rDNA followed by primer extension analysis showed that overexpression of USF1 and USF2 as homodimers resulted in inhibition of rDNA transcription by as much as 85 – 90% whereas overexpression of USF1/USF2 in the heterodimeric form activated transcription approximately 3.5-fold. Transfection of mutant USF2 cDNA that is devoid of the basic DNA-binding domain produced only minimal inhibition of rDNA transcription. These data show that USF can modulate transcription of rRNA gene in vivo by functioning as a repressor (homodimer) or activator (heterodimer) of pol I transcription in vivo and suggest that inhibition of rDNA transcription may be responsible for the antiproliferative action of USF homodimers.

The transcription factor GATA3 is a key regulator of mammary gland development and a definitive marker of luminal breast cancer. However, the molecular mechanisms underlying the role of GATA3 in breast carcinogenesis is still not fully understood. We report here that GATA3 promotes cell proliferation and tumorigenesis by facilitating the G1/S transition through its transcription regulation of the CCND1 gene in breast cancer cells. We found that GATA3 is physically associated with poly-ADP ribose polymerase-1 (PARP1), an enzyme modifying nuclear proteins by poly(ADP-ribosyl)ation. We showed that PARP1 acts as a transcription coactivator for GATA3 in breast cancer cells and demonstrated that GATA3 cooperates with PARP1 in transactivation of the CCND1 gene. We demonstrated that PARP1 competes with linker histone H1 to maintain a transcriptional competent chromatin environment for CCND1 gene. Our results unveiled a molecular basis for the coordinated regulation between GATA3 and PARP1 in transcription activation, providing a mechanism for GATA3 in breast carcinogenesis.

Disruption of the circadian rhythm is now believed to associate with a number of hormone-related cancers, such as breast cancer, in which aberrant estrogen receptor-α (ERα) signaling is a major contributor. However, the molecular mechanisms underlying the function of core clock proteins in cancer are still largely undefined. In this study, we showed that circadian locomotor output cycles kaput (CLOCK), a key circadian protein, can interact with ERα. Furthermore, this interaction was enhanced by estrogen. We also showed that CLOCK can be sumoylated and sumoylation of CLOCK, which is also stimulated by estrogen, had two consequences: (1) it increased the transcriptional activity of CLOCK; and (2) it increased the CLOCK-modulated transcriptional activity of ERα, as shown by increased transcription of cyclin D1. Sumoylation of CLOCK occurred at two lysine residues, K67 and K851. The enhancement of ERα transcriptional activity exerted by wild-type but not mutant (2K/2R) CLOCK in response to estrogen indicated that sumoylation of CLOCK may have an important role in estrogen-dependent signaling. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay conducted with breast cancer cell lines (MCF-7 and T47D) demonstrated that sumoylation of CLOCK stimulated cell growth and increased the proportion of S phase cells in the cell cycle. The results of this study uncovered new insight into the connection between a major circadian protein and a major estrogen-dependent transcription factor, providing the basis for further research into the involvement of circadian proteins in breast cancer.

15-Lipoxygenase-1 (15-LOX-1) is transcriptionally silenced in cancer cells, and its transcription reactivation (for example, through histone deacetylase inhibitors (HDACIs)) restores apoptosis to cancer cells. However, the exact mechanism underlying 15-LOX-1 transcription reactivation in cancer cells is still undefined. Therefore, we evaluated the critical mechanisms required for 15-LOX-1 transcription reactivation in colon cancer cells. Specific HDAC1 and HDAC2 inhibition activated 15-LOX-1 transcription. 15-LOX-1 transcription was repressed through transcription repressor complex recruitment in the region of -120 to -391 of the 15-LOX-1 promoter. The nucleosome remodeling and histone deacetylase (NuRD) repression complex was recruited to this region. Depsipeptide significantly reduced the recruitment of NuRD key components (for example, metastasis-associated protein 1 (MTA1) and HDAC1) to the 15-LOX-1 promoter before 15-LOX-1 transcriptional activation. Knock down of NuRD key components (for example, MTA1 and HDAC1) by small interfering RNA (siRNA) activated 15-LOX-1 transcription, as measured by luciferase reporter assays in stably transfected SW480 cells with the 15-LOX-1 promoter construct of the -391, but not the -120 region. Relative to expression in normal tissue, MTA1 expression in colorectal cancer mucosa from colorectal cancer patients was negatively related to 15-LOX-1 expression. Thus, our results show that NuRD contributes to 15-LOX-1 transcription suppression in colon cancer cells and that HDACIs can inhibit NuRD recruitment to a promoter to activate gene transcription, as in the case of 15-LOX-1.

The most frequent targets of genetic alterations in human lymphoid leukemias are transcription factor genes with essential functions in blood cell development. TAL1, LYL1, HOX11 and other transcription factors essential for normal hematopoiesis are often misexpressed in the thymus in T-cell acute lymphoblastic leukemia (T-ALL), leading to differentiation arrest and cell transformation. Recent advances in the ability to assess DNA copy number have led to the discovery that the MYB transcription factor oncogene is tandemly duplicated in T-ALL. The NOTCH1 gene, which is essential for key embryonic cell-fate decisions in multicellular organisms, was found to be activated by mutation in a large percentage of T-ALL patients. The gene encoding the FBW7 protein ubiquitin ligase, which regulates the turnover of the intracellular form of NOTCH (ICN), is also mutated in T-ALL, resulting in stabilization of the ICN and activation of the NOTCH signaling pathway. In mature B-lineage ALL and Burkitt lymphoma, the MYC transcription factor oncogene is overexpressed due to translocation into the IG locus. PAX5, a transcription factor essential for B-lineage commitment, is inactivated in 32% of cases of B-progenitor ALL. Translocations resulting in oncogenic fusion transcription factors also occur frequently in this form of ALL. The most frequent transcription factor chimeric fusion, TEL-AML1, is an initiating event in B-progenitor ALL that acts by repressing transcription. Therefore, deregulated transcription and its consequent effects on key developmental pathways play a major role in the molecular pathogenesis of lymphoid malignancy. Once the full complement of cooperating mutations in transformed B- and T-progenitor cells is known, and the deregulated downstream pathways have been elucidated, it will be possible to identify vulnerable components and to target them with small-molecule inhibitors.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.

Basic helix-loop-helix (bHLH) transcription factors play a pivotal role in the regulation of tumorigenesis, and also in a wide range of other developmental processes in diverse species from yeast to humans. Here we demonstrate for the first time that Ret finger protein (RFP), a member of the TRIM family of proteins initially identified as a recombined transforming gene from a human lymphoma, is a novel interaction partner for four different bHLH proteins (SCL, E47, MyoD and mASH-1), but does not interact with GATA-1 or PU.1. Interaction with SCL required the B-box and first coiled-coil region of RFP together with the bHLH domain of SCL. RFP was able to repress transcriptional activation by E47, MyoD and mASH-1, but not by members of several other transcription factor families. Transcriptional repression by RFP was trichostatin A sensitive and did not involve an Id-like mechanism or ubiquitination with subsequent degradation of bHLH proteins. Instead, our results suggest that bHLH transcription factors are regulated by a previously undescribed interaction with RFP, which functions to recruit HDAC and/or Polycomb proteins and thus repress target genes of bHLH proteins. These results reveal an unexpected link between the bHLH and TRIM protein families.

In the budding yeast, Saccharomyces cerevisiae, a significant fraction of genes (10%) are transcribed with cell cycle periodicity. These genes encode critical cell cycle regulators as well as proteins with no direct connection to cell cycle functions. Cell cycle-regulated genes can be organized into 'clusters' exhibiting similar patterns of regulation. In most cases periodic transcription is achieved via both repressive and activating mechanisms. Fine-tuning appears to have evolved by the juxtaposition of regulatory motifs characteristic of more than one cluster within the same promoter. Recent reports have provided significant new insight into the role of the cyclin-dependent kinase Cdk1 (Cdc28) in coordination of transcription with cell cycle events. In early G1, the transcription factor complex known as SBF is maintained in a repressed state by association of the Whi5 protein. Phosphorylation of Whi5 by Cdk1 in late G1 leads to dissociation from SBF and transcriptional derepression. G2/M-specific transcription is achieved by converting the repressor Fkh2 into an activator. Fkh2 serves as a repressor during most of the cell cycle. However, phosphorylation of a cofactor, Ndd1, by Cdk1 late in the cell cycle promotes binding to Fkh2 and conversion into a transcriptional activator. Such insights derived from analysis of specific genes when combined with genome-wide analysis provide a more detailed and integrated view of cell cycle-dependent transcription.

USF and c-Myc are basic helix-loop-helix transcription factors with similar DNA-binding specificities, but antagonistic effects on cellular transformation. In order to determine how these opposite functions correlate with the transcriptional activities of the two factors on particular downstream targets, we investigated the roles of USF and c-Myc in expression of CDK4, a known direct target of c-Myc. Overexpression of either c-Myc or USF2, but not USF1, stimulated the expression of CDK4 promoter-driven reporter genes in the non-tumorigenic mammary epithelial MCF-10A cells. Dominant-negative mutants specific to either Myc or USF family proteins inhibited reporter gene activity as well as endogenous CDK4 expression, demonstrating involvement of both USF and Myc in CDK4 transcriptional control. In contrast, in two different breast cancer cell lines where USF is transcriptionally inactive and c-Myc is overexpressed, CDK4 promoter activity was no longer responsive to either transcription factor. Accordingly, chromatin immunoprecipitation revealed significantly lower levels of both USF and c-Myc bound to the endogenous CDK4 promoter in breast cancer cells than in MCF-10A cells, with a concomitant decrease in associated histone H3 acetylation. These results suggest that a major switch in the transcriptional control of CDK4 occurs during breast carcinogenesis, with likely alteration of cell cycle regulation.

MCM10 and TopBP1 function in the initiation of DNA replication, by regulating the chromatin binding of the DNA polymerase alpha loading factor, CDC45. TopBP1 is also known as a DNA damage response protein. In this study, we showed that the transcription of human MCM10 and TopBP1 is activated by transcription factors E2F1-3, but not by factors E2F4-7. Analysis of various MCM10 and TopBP1 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1-induced activation of MCM10 and TopBP1 gene transcription, which is further suppressed by pRb. The promoter activities of human MCM10 and TopBP1 were demonstrated to be growth dependent via the E2F-responsive sequence. Although E2F1 was stabilized by ultraviolet (UV) irradiation, the mRNA expression level of TopBP1 was suppressed in HCT116 human diploid colon cancer cells. We showed, by performing chromatin immunoprecipitation that, in response to UV irradiation but not doxorubicin treatment, E2F4 accumulated on the MCM10 and TopBP1 promoters. Our data suggest a model in which UV irradiation-induced DNA damage depends, at least in part, on the accumulation of the E2F4 transcription factor on the MCM10 and TopBP1 promoters, which results in suppression of DNA replication.

Basal transcription of the HIV-1 genome is controlled by a variety of ubiquitous and inducible regulatory factors, some with the ability to associate with the viral DNA sequences within the promoter spanning the long terminal repeat (LTR). In this report we demonstrate that activation of the HIV-1 promoter through the inducible DNA binding NF-kappaB transcription factors can be affected by cdk9 in human astrocytic cells. Our results show that ectopic expression of cdk9, but not its mutant variant which lacks the domain responsible for its kinase activity, augments transcription of the LTR. Moreover, we demonstrate that induction of the NF-kappaB pathway by PMA, or overexpression of its subunits including p50/p65 have a negative effect on the ability of cdk9 to stimulate viral gene transcription in these cells. Results from band-shift experiments demonstrated significant suppression of p50/p65 association to its DNA target motif by cdk9. Further, data from GST pull-down and combined immunoprecipitation/Western blot analysis of the protein extracts from cells expressing cdk9, p50 and p65 have revealed the interaction of cdk9 with both p50 and p65 in the absence of DNA containing the kappaB motif. All of these observations led us to conclude that the interaction of cdk9 with the NF-kappaB factors can determine the ability of NF-kappaB to modulate HIV-1 gene transcription.

Transcription of the ERBB2 oncogene is repressed by oestrogen in human breast cancer cells. We show that a 218 bp fragment of the human ERBB2 gene promoter is responsive to oestrogen in transient transfection in ZR75.1 and SKBR.3 cells when the oestrogen receptor is expressed. Deletion analysis of this fragment shows that a sequence located at the 5' end, which is known to mediate ERBB2 overexpression in breast cancer, is also responsible for the oestrogen response. This sequence binds AP-2 transcription factors and appears functionally identical to an element of the oestrogen-dependent enhancer described in the first intron of human ERBB2. We observed that oestrogen treatment down-regulates expression of AP-2 proteins but does not affect the DNA binding activity of AP-2. Constitutive expression of AP-2beta or AP-2gamma, but not AP-2alpha, abrogates the estrogenic repression. Our results demonstrate that AP-2 transcription factors are implicated in the oestrogenic regulation of ERBB2 gene expression and suggest a complex interplay involving the different AP-2 isoforms and other unidentified factors.

Although USF-1 and -2 are the major proteins that bind to Myc-regulated E-box (CACGTG) elements in many cells, there is no clear role for USF during Myc-dependent gene regulation. Using dominant negative alleles of USF-1 we now show that DNA binding by USF at a Myc-regulated E-box limits the ability of another E-box binding factor, TFE-3, to activate a target gene of Myc in vivo and to stimulate S phase entry in resting fibroblasts. Similarly, dominant negative alleles of USF-1 relieve the restriction that prevents activation of the IgH enhancer by TFE-3 in non B-cells. DNA binding activity of USF complexes is abundant in primary human B-cells and is significantly downregulated during B-cell immortalization. Re-expression of USF-1 in immortalized B-cells retards proliferation. Our data establish an essential role for USF in restricting E-box dependent gene activation in vivo and suggest that this control is relaxed during cellular immortalization.

Activation of the TAL1 (or SCL) gene, originally identified through its involvement by a recurrent chromosomal translocation, is the most frequent gain-of-function mutation recognized in T-cell acute lymphoblastic leukemia (T-ALL). The TAL1 proteins contain a basic helix - loop - helix (bHLH) motif characteristic of a large family of transcription factors that control transcription from an E box target element as heterodimers with the E2A- and HEB-encoded gene products. Gene knockout studies in mice indicate that this transcription factor is required for embryonic and adult hematopoiesis, and considerable evidence suggests it has specific functions in terminal erythroid differentiation. We investigated whether the broadly expressed nuclear protein p300, known to function as a coactivator for other bHLH proteins involved in cellular differentiation, also interacts with TAL1. p300 was found to coimmunoprecipitate with Tal1 in extracts from murine erythroleukemia (MEL) cells induced to differentiate with dimethylsulfoxide (DMSO), and p300 and Tal1 were observed in a common E box DNA-binding complex in extracts from differentiating MEL cells. p300 also interacted with Tal1 in protein pulldown assays, suggesting this was a direct interaction. Finally, p300 augmented transcription by Tal1 from an E box-containing promoter and by a GAL4-Tal1 fusion from a promoter containing the GAL4 DNA-binding element. Deletion analysis identified the bHLH domain of Tal1 and amino-terminal sequences of p300 as necessary for p300-stimulated transactivation and Tal1-p300 interaction in vitro. These results indicate that recruitment of the transcriptional coactivator p300 can positively regulate TAL1-directed gene expression. The dependence of their interaction in MEL cells on addition of a differentiation inducer suggests, further, that this TAL1-p300 complex may have an important role in terminal erythroid differentiation.

A large body of evidence has been accumulated that demonstrates dominant effects of Myc proto-oncoproteins on different aspects of cellular growth. Myc is one of the few proteins that is sufficient to drive resting cells into the cell cycle and promote DNA synthesis. In line with this finding is that the constitutive expression of Myc in cells blocks their differentiation. These growth stimulating properties are most likely responsible for Myc's ability to initiate and promote tumor formation. Interestingly Myc can also sensitize cells to apoptosis, suggesting that this protein is part of a life-and-death switch. Molecularly Myc functions as a transcriptional regulator that needs to heterodimerize with Max to exert the biological activities described above and to regulate gene transcription. Myc and Max are just two members of a growing family of proteins referred to as the Myc/Max/Mad network. A hallmark of these proteins is that they possess a C-terminal basic region/helix-loop-helix/leucine zipper domain (bHLHZip). The bHLHZip domain specifies dimerization within the network and determines sequence specific DNA binding. Importantly this domain together with the N-terminal transactivation domain is essential for Myc biology. Here we have summarized the structural, functional, and regulatory aspects of the bHLHZip domain of Myc proteins.

dMax, a naturally occurring splice variant of the Myc binding protein Max, lacks the DNA binding basic region and helix 1 of the Helix – Loop – Helix domain; dMax interacts with c-Myc in vitro and in vivo, and inhibits E-box Myc site driven transcription in transient transfection assays. Here we have investigated the expression, function and interactions of dMax. RT/PCR analyses detected dmax mRNA in multiple tissues of the developing, newborn and adult mouse. Functionally, dMax reduced the ability of c-Myc to cooperate with the progression factor A-Myb to promote S phase entry of quiescent smooth muscle cells. In contrast, dMax failed to ablate inhibition of initiator element (Inr)-mediated transcription by c-Myc in Jurkat T cells. In in vitro protein : protein association assays, dMax interacted with c-Myc, N-Myc, L-Myc, Mad1, Mxi1, Mad3 and Mad4, but not with itself or wild-type Max. These interactions required an intact leucine zipper. Inhibition of E-box-mediated transactivation by induction of dMax overexpression resulted in apoptosis of WEHI 231 B cells. Thus, dMax is a widely expressed, naturally occurring protein, with the capacity to bind most members of the Myc/Max superfamily; dMax has little effect on Inr-mediated repression by c-Myc, but can significantly decrease E-box-mediated events promoting proliferation and cell survival.