We have determined that the developmental control of immunoglobulin K 3' enhancer (cE3') activity is the result ofthe combined influence of positive-and negative-acting elements. We show that a central core in the dcE3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements. The negative-acting sequences repress enhancer activity in a position-and orientationindependent manner at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to the negative-acting segment of the KE3' enhancer. This protein also binds to the immunoglobulin heavy-chain enhancer ,uE1 site. NF-E1 is encoded by the same gene as the YY-1 protein, which binds to the adeno-associated virus P5 promoter. NF-E1 is also the human homologue of the mouse 8 protein, which binds to ribosomal protein gene promoters. The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors. Cotransfection studies with this cDNA indicate that it can repress basal promoter activity. The apparent dual function of this protein is discussed.

Polycomb group (PcG) proteins are responsible for maintaining transcriptional repression of developmentally important genes. However, the mechanism of PcG recruitment to specific DNA sequences is poorly understood. Transcription factor YY1 is one of the few PcG proteins with sequence-specific DNA binding activity. We previously showed that YY1 can recruit other PcG proteins to DNA, leading to histone posttranslational modifications and stable transcriptional repression. Using Drosophila transgenic approaches, we identified YY1 sequences 201-226 as necessary and sufficient for PcG transcriptional repression in vivo. When fused to a heterologous DNA-binding domain, this short 26-aa motif was sufficient for transcriptional repression, recruitment of PcG proteins to DNA, and methylation of histone H3 lysine 27. Deletion of this short YY1 motif did not affect transient transcriptional repression but ablated PcG repression, PcG protein recruitment to DNA, and methylation of H3 lysine 27. We propose that this motif be named the REPO domain for its function in recruitment of Polycomb. The REPO domain is well conserved in YY1 orthologs and in related proteins.repression ͉ transcription ͉ chromatin P olycomb group (PcG) proteins are responsible for the heritable silencing of target genes in metazoans (1, 2) and are functionally conserved in Drosophila and mammals (2, 3). Mutations in PcG genes result in misexpression of target genes with resulting homeotic transformation of body parts (4). Studies in mammals showed that PcG proteins are important for normal skeletal, muscular, and hematopoietic development (5). A variety of studies support the existence of at least two PcG complexes, each composed of several polypeptides: PRC1, containing Polycomb (Pc), polyhomeotic (Ph), dRing, and posterior sex combs (Psc); and PRC2 containing enhancer of zeste [E(z)], extra sex combs (Esc), and suppressor of zeste 12 [Su(z)12] (1, 2). These complexes are recruited to chromatin, where they maintain transcriptional silencing. An unresolved issue is the mechanism of recruitment of PcG complexes to appropriate target genes.The mechanism of PcG complex recruitment has remained elusive, especially in mammals. Drosophila Polycomb response elements (PREs) bind to PcG proteins and silence cis-linked genes in vivo (1, 2). The large size and poor sequence homology of characterized PREs has hampered the search for recruiting factors. Furthermore, nearly all characterized PcG proteins lack sequencespecific DNA-binding activity. Exceptions include the Drosophila PcG proteins Pleiohomeotic (PHO) and Pleiohomeotic-like (PHOL) and their mammalian counterpart, the vertebrate transcription factor Yin Yang 1 (YY1) (6, 7). Homology between these transcription factors is localized to the C-terminal zinc-finger DNA-binding domain (YY1 residues 298-414, 95% identical to PHO) and a short internal sequence (YY1 residues 205-226, 82% identical to PHO). We previously demonstrated that, similar to PHO, YY1 can function as a PcG protein to mediate silencing (8). Th...

The Wnt/b-catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt-induced stabilization of the b-catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real-time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated b-catenin-responsive luciferase activity in a dose-dependent manner and decreased the b-catenin levels. By utilizing a yeast two-hybrid screen, bTrCP, a negative regulator of b-catenin was identified as a novel Dkk3-interacting partner. Coexpression with bTrCP synergistically enhanced the inhibitory function of Dkk3 on b-catenin. The stable expression of Dkk3 blocks the nuclear translocation of b-catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of b-catenin and its downregulation contribute to an activation of the b-catenin signaling pathway.

YY1 is a multifunctional transcription factor capable of either activation or repression of transcription. Using a series of mutant proteins, we have characterized domains responsible for activation or repression. We found that the YY1 transcriptional activation domain lies near the amino terminus and requires amino acids 16 -29 and 80 -100 for maximal activity. The region between residues 16 and 29 has the potential to form an acidic amphipathic helix, whereas residues between 80 and 100 are rich in proline and glutamine. The YY1 repression domain lies near the carboxyl terminus and is embedded within the YY1 zinc finger region necessary for binding to DNA. Deletion of YY1 amino acids, which include zinc fingers 3 and 4, abolishes repression. However, site-directed mutagenesis, progressive deletion, and internal deletion mutant analyses indicate that the normal structures of zinc fingers 3 and 4 are not required for repression.YY1 (variously called NF-E1, ␦, or UCRBP; Refs. 1-4), is a multifunctional transcription factor that can either activate or repress transcription. Repression has been observed in the context of the immunoglobulin 3Ј enhancer, the Moloney murine leukemia virus long terminal repeat, the adeno-associated virus P5 promoter, the skeletal ␣-actin promoter, the ␤-casein promoter, ⑀-and ␥-globin genes, the serum amyloid A1 promoter, the human immunodeficiency virus promoter, and the human papilloma virus type 18 promoter (1, 4 -13; reviewed in ref. 14). In contrast YY1 can activate the c-Myc promoter, the ribosomal protein L30 and L32 promoters, and the intracisternal A-particle upstream promoter element (15-18). Interestingly, YY1 can either activate or repress some promoters depending upon either promoter architecture or intracellular milieu. For instance, YY1 typically represses the adeno-associated virus P5 promoter, but can be converted into a potent transcriptional activator in the presence of adenovirus E1A protein (1). YY1 can also either activate or repress the c-Fos promoter based on the orientation or the position of a YY1 binding site within the promoter (19). Finally, YY1 can either repress or activate the human papilloma virus type 18 promoter depending upon the presence of an adjacent DNA sequence that binds to a distinct nuclear factor (6).The mechanism of YY1 function is presently unclear. In some cases, YY1 binding appears to preclude the binding of activator proteins. For instance, YY1 binding competes with the binding of NF-B to an overlapping -sequence in the serum amyloid A1 promoter (13). Similarly, YY1 competes with serum response factor binding in the ␣-actin promoter, GATA-1 binding in the ⑀-globin promoter, and with binding of a lactation-associated factor in the ␤-casein promoter (7, 9 -11). YY1 function may also relate to its ability to bend DNA (19). In other cases, the function of YY1 appears to be controlled by interaction with other proteins such as adenoviral E1A, c-Myc, or switch binding protein (1,6,20). Interestingly, loss of YY1 binding sites in the human pap...

SUMOylation of transcription factors often attenuates transcription activity. This regulation of protein activity allows more diversity in the control of gene expression. Interferon regulatory factor-1 (IRF-1) was originally identified as a regulator of IFN-␣/␤, and its expression is induced by viral infection or IFN stimulation. Accumulating evidence supports the theory that IRF-1 functions as a tumor suppressor and represses the transformed phenotype. Here we report that the level of SUMOylated IRF-1 is elevated in tumors. Site-directed mutagenesis experiments disclose that the SUMOylation sites of IRF-1 are identical to the major ubiquitination sites. Consequently, SUMOylated IRF-1 displays enhanced resistance to degradation. SUMOylation of IRF-1 attenuates its transcription activity, and SUMOylated IRF-1 inhibits apoptosis by repression of its transcriptional activity. These data support a mechanism whereby SUMOylation of IRF-1 inactivates its tumor suppressor function, which facilitates resistance to the immune response.tumor suppressor ͉ Ubc9 ͉ SENP1

No abstract

During carcinogenesis, NF-κB mediates processes associated with deregulation of the normal control of proliferation, angiogenesis, and metastasis. Thus, suppression of NF-κB has been linked with chemoprevention of cancer. Accumulating findings reveal that heat shock protein 90 (HSP90) is a molecular chaperone and a component of the IκB kinase (IKK) complex that plays a central role in NF-κB activation. HSP90 also stabilizes key proteins involved in cell cycle control and apoptosis signaling. We have determined whether the exogenous administration of isoflavone-deprived soy peptide prevents 7,12-dimethylbenz[α]anthracene (DMBA)-induced rat mammary tumorigenesis and investigated the mechanism of action. Dietary administration of soy peptide (3.3 g/rat/day) significantly reduced the incidence of ductal carcinomas (50%), the number of tumors per multiple tumor-bearing rats (49%; P ＜ 0.05), and extended the latency period of tumor development (8.07 ± 0.92 weeks) compared to control diet animals (10.80 ± 1.30; P ＜ 0.05). Our results have further demonstrated that soy peptide (1) dramatically inhibits the expression of HSP90, thereby suppressing signaling pathway leading to NF-κB activation; (2) induces expression of p21, p53, and caspase-3 proteins; and (3) inhibits expression of VEGF. In agreement with our in vivo data, soy peptide treatment inhibited the growth of human breast MCF-7 tumor cells in a dose-dependent manner and induced apoptosis. Taken together, our in vivo and in vitro results suggest chemopreventive and tumor suppressive functions of isoflavone-deprived soy peptide by inducing growth arrest and apoptosis.

Promyelocytic leukemia zinc finger protein (PLZF) is a sequence-specific, DNA binding, transcriptional repressor differentially expressed during embryogenesis and in adult tissues. PLZF is known to be a negative regulator of cell cycle progression. We used PLZF as bait in a yeast two-hybrid screen with a cDNA library from the human ovary tissue. A novel cervical cancer suppressor 3 (CCS-3) was identified as a PLZF interacting partner. Further characterization revealed the BTB domain as an interacting domain of PLZF. Interaction of CCS-3 with PLZF in mammalian cells was also confirmed by co-immunoprecipitation and in vitro binding assays. It was found that, although CCS-3 shares similar homology with eEF1A, the study determined CCS-3 to be an isoform. CCS-3 was observed to be downregulated in human cervical cell lines as well as in cervical tumors when compared to those from normal tissues. Overexpression of CCS-3 in human cervical cell lines inhibits cell growth by inducing apoptosis and suppressing human cyclin A2 promoter activity. These combined results suggest that the potential tumor suppressor activity of CCS-3 may be mediated by its interaction with PLZF.