The human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter binds a sequence-specific single-strand DNA binding protein termed NF-GMb. We previously demonstrated that the NF-GMb binding sites were required for repression of tumor necrosis factor-alpha (TNF-alpha) induction of the proximal GM-CSF promoter sequences in fibroblasts. We now describe the isolation of two different cDNA clones that encode cold shock domain (CSD) proteins with NF-GMb binding characteristics. One is identical to the previously reported CSD protein dbpB and the other is a previously unreported variant of the dbpA CSD factor. This is the first report of CSD factors binding to a cytokine gene. Nuclear NF-GMb and expressed CSD proteins have the same binding specificity for the GM-CSF promoter and other CSD binding sites. We present evidence that CSD factors are components of the nuclear NF-GMb complex. We also demonstrate that overexpression of the CSD proteins leads to complete repression of the proximal GM-CSF promoter containing the NF-GMb/CSD binding sites. Surprisingly, we show that CSD overexpression can also directly repress a region of the promoter which apparently lacks NF-GMb/CSD binding sites. NF-GMb/CSD factors may hence be acting by two different mechanisms. We discuss the potential importance of CSD factors in maintaining strict regulation of the GM-CSF gene.
NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.
The tumor necrosis factor-␣-responsive region of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter (؊114 to ؊31) encompasses binding sites for NF-B, CBF, AP-1, ETS, and NFAT families of transcription factors. We show both here and previously that mutation of any one of these binding sites greatly reduces tumor necrosis factor-␣ induction of the GM-CSF promoter. Interspersed between these elements are sequences that when mutated lead to an increase in GM-CSF promoter activity. We have previously shown that two of these repressor elements bind proteins known as cold shock domain (CSD) factors and that overexpression of CSD proteins leads to repression of GM-CSF promoter activity in fibroblasts. CSD proteins are single strand DNA-and RNA-binding proteins that contact 5-CCTG-3 sequences in the GM-CSF repressor elements. We show here that two newly identified repressor sequences in the proximal promoter can also bind CSD proteins. We have characterized the CSDcontaining protein complexes that bind to the GM-CSF promoter and identified a novel protein related to mitochondrial single strand binding protein that forms part of one of these complexes. The four CSD-binding sites on the promoter occur in pairs on opposite strands of the DNA and appear to form an ordered array of binding elements. A similar ordered array of CSD sites are present in the promoters of the granulocyte colony-stimulating factor and interleukin-3 genes, implying a common mechanism for negative regulation of these myeloid growth factors.
To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the selfassociating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors. Despite their similarity in sequence and structure IGF-I and IGF-II can stimulate both overlapping and distinct biological functions (reviewed in Ref. 9). This is evident in patients with IGF-I deficiency, which results in severe growth and mental retardation, where IGF-II does not compensate for the loss of IGF-I activity (10 -12). Therefore, in order to understand how both IGF-I and IGF-II stimulate their respective biological outcomes, we first need to understand the mechanism by which both ligands bind and in turn activate the IGF-1R. Insulin-like growth factors (IGFsThe IGF-1R is a member of the tyrosine kinase family of receptors and, together with the insulin receptor (IR) and insulin-related receptor, forms a subfamily with similar structural organization (reviewed in Ref. 9). The IGF-1R and IR are homodimers composed of two ␣ and two  subunits and are synthesized as a single precursor polypeptide, which is then post-translationally processed by dimerization, proteolytic cleavage, and glycosylation.The amino-terminal regions of the ␣ chain of these receptors are composed of three domains, two structurally homologous subdomains designated large homologous domain 1 (L1) and large homologous domain 2 (L2), which are separated by a cysteine-rich (CR) domain of ϳ160 amino acids (3) (see supplemental Fig. S1). The major ligand binding determinants of the structurally related IGF-1R and IR reside within the extracellular ␣ subunits of the receptors (reviewed in Refs. 9, 13, and 14). Studies performed with truncated IRs have demonstrated that dimeriza...
A conserved DNA sequence element, termed cytokine 1 (CK-1), is found in the promoter regions of many hemopoietic growth factor (HGF) genes. Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor, NF-GMa, which is a protein with a molecular mass of 43 kilodaltons. It interacts with different affinities with the CK-1-like sequence from a number of HGF genes, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF, interleukin 3 (IL-3), and IL-5. We show here that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor-a (TNF-a) treatment and that the CK-1 sequence from the G-CSF gene is a TNF-a-responsive enhancer in these cells. The NF-GMa protein is distinct from another TNF-oa-responsive transcription factor, NF-KB, by several criteria. Firstly, several NF-KcB-binding sites, although having sequence similarity with the CK-1 sequence, cannot compete efficiently for NF-GMa binding to CK-1. Secondly, the CK-1 sequence from both G-CSF and GM-CSF does not respond to phorbol ester treatment as would an NF-KB-binding element. These results demonstrate that NF-GMa is a novel transcription factor inducible by TNF-a and binds to a common element in HGF gene promoters.
The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tatinteracting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tatdependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery. The human immunodeficiency virus (HIV)1 transactivator Tat is an 86 -101-amino acid protein that is a potent transcriptional activator of expression of all HIV proteins, including Tat itself. Tat expression is essential for HIV viral replication, because Tat defective viruses are unable to replicate or express viral proteins efficiently (1, 2). The Tat-responsive region of the HIV LTR has been mapped to an element immediately downstream of the transcription start site (3, 4). Tat is unusual in that it is targetted to the promoter by binding to a 59-nucleotide stem-loop structure in the nascent RNA called the transactivation-responsive (TAR) element, which is found at the 5Ј end of all HIV viral RNAs (5-8). Although Tat has been demonstrated to have effects on transcriptional initiation (9), it differs from conventional activators in that its primary effect appears to be on transcriptional elongation (9 -11). In the absence of Tat, short prematurely terminated RNA transcripts are produced, but Tat appears to modify RNA polymerase II to a form that elongates more efficiently (12).Recently a mechanism has been established to explain how Tat promotes transcriptional elongation. Phosphorylation of the C-terminal domain of RNA polymerase II by cellular kinases marks the transition from an initiation complex to an efficiently elongating polymerase complex, and a primary role of Tat appears to be to recruit such kinases. Tat targets two cyclin-dependent kinases, cdk7, the kinase component of TFIIH, and cdk9, the kinase component of positive transcription elongation factor b (pTEFb), which together are proposed to hyperphosphorylate RNA polymerase II (reviewed in Refs. 13 and 14). Tat, through its ac...
Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.
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