The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1.

Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling.

Signals emanating from CD40 play crucial roles in B-cell function. To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to done cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned. TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor. The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1. In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2. Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5. In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor kappa B. Furthermore, amino-terminally truncated forms of TRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF3. These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.

Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

Identification of potential target genes for the neuron-restrictive silencer factor.

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.

Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-kappa B in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-kappa B by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose- and time-dependent manner. Besides TNF, CAPE also inhibited NF-kappa B activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-kappa B activation. CAPE prevented the translocation of the p65 subunit of NF-kappa B to the nucleus and had no significant effect on TNF-induced I kappa B alpha degradation, but did delay I kappa B alpha resynthesis. The effect of CAPE on inhibition of NF-kappa B binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-1, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-kappa B activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.