Signal transducer and activator of transcription 3 (Stat3) is a key regulator of gene expression in response to signaling of the glycoprotein 130 (gp130) family cytokines, including interleukin 6, oncostatin M, and leukemia inhibitory factor. Many efforts have been made to identify Stat3 target genes and to understand the mechanism of how Stat3 regulates gene expression. Using the microarray technique, hundreds of genes have been documented to be potential Stat3 target genes in different cell types. However, only a small fraction of these genes have been proven to be true direct Stat3 target genes. Here we report the identification of novel direct Stat3 target genes using a genome-wide screening procedure based on the chromatin immunoprecipitation method. These novel Stat3 target genes are involved in a diverse array of biological processes such as oncogenesis, cell growth, and differentiation. We show that Stat3 can act as both a repressor and activator on its direct target genes. We further show that most of the novel Stat3 direct target genes are dependent on Stat3 for their transcriptional regulation. In addition, using a physiological cell system, we demonstrate that Stat3 is required for the transcriptional regulation of two of the newly identified direct Stat3 target genes important for muscle differentiation.The signal transducer and activator of transcription (STAT) 2 family of transcriptional regulators is activated in response to extracellular signaling proteins, including cytokines and growth factors (1, 2). When cytokines bind to their cell surface receptors, the receptor-associated JAK tyrosine kinases become activated and in turn phosphorylate a single tyrosine residue in the STAT molecule. The phosphorylated STATs then enter the nucleus as dimers and bind to specific DNA sequences in the promoters of their target genes to regulate transcription. Although the seven members of the STAT family have similarity in their molecular structure and function, they play diverse physiological roles in a wide variety of biological processes (3).One member of the STAT family, Stat3, mediates the signaling of cytokines that share the gp130 receptor chain, which include interleukin-6, oncostatin M (OSM), and leukemia inhibitory factor (LIF) (4, 5). In response to gp130 ligand stimulation, Stat3 is phosphorylated on Tyr-705 and forms dimers through phosphotyrosine-Src homology 2 domain interactions (6). The dimerized Stat3 molecules enter the nucleus and bind to a consensus DNA sequence in the promoters of its target genes to regulate transcription (7). The transcriptional activity of Stat3 is mediated by its transcription activation domain located in the carboxyl-terminal end of the molecule (8). In addition to the tyrosine phosphorylation, the Stat3 transcription activation domain contains a serine residue (Ser-724) that is also phosphorylated to achieve maximum transcription activity (9, 10). Analyses of Stat3-dependent enhancersomes demonstrate that Stat3 interacts and recruits other transcription factors and ...

Background:The goal is to understand the molecular mechanism of metastasis and the roles of IL-6/OSM, STAT3, and fascin. Results: STAT3 binds to the fascin promoter and is essential for its expression and cell migration in response to IL-6/OSM. Conclusion: STAT3 plays a central role in cell migration through direct control of fascin expression. Significance: Drug targets are identified to block tumor metastasis.

The eukaryotic minichromosome maintenance (MCM) family of proteins (MCM2-MCM7) is evolutionarily conserved from yeast to human. These proteins are essential for DNA replication. The signal transducer and activator of transcription proteins are critical for the signal transduction of a multitude of cytokines and growth factors leading to the regulation of gene expression. We previously identified a strong interaction between Stat1 and MCM5. However, the physiological significance of this interaction was not clear. We show here by chromatin immunoprecipitation (ChIP) analyses that the MCM5 protein, as well as other members of the MCM family, is inducibly recruited to Stat1 target gene promoters in response to cytokine stimulation. Furthermore, the MCM proteins are shown to move along with the RNA polymerase II during transcription elongation. We have also identified an independent domain in MCM5 that mediates the interaction between Stat1 and MCM5; overexpression of this domain can disrupt the interaction between Stat1 and MCM5 and inhibit Stat1 transcriptional activity. Finally, we used the RNA interference technique to show that MCM5 is essential for transcription activation of Stat1 target genes. Together, these results demonstrate that, in addition to their roles in DNA replication, the MCM proteins are also necessary for transcription activation.RNA polymerase II ͉ DNA helicase ͉ IFN-␥ T he evolutionarily conserved eukaryotic minichromosome maintenance (MCM) family of proteins consists of six members: MCM2-MCM7 (reviewed in refs. 1 and 2). The molecular structure and in vitro analyses of these proteins suggest that they function as a DNA helicase (3); they form a heterohexamer complex that binds to DNA replication origins and moves along with the DNA polymerase during DNA replication elongation (4, 5). In addition to the hexamer complex, the MCM proteins also form subcomplexes containing some members of the family, such as MCM4͞6͞7 or MCM3͞5 (3,[6][7][8]. It has been suggested that these subcomplexes represent segments during the assembly of the hexameric MCM complex (9). The MCM proteins are also highly abundant, and their number far exceeds that of the replication origins in yeast (8,(10)(11)(12). These observations have led to the suggestion that the MCM proteins may play additional roles in other biological processes, such as DNA repair, chromatin remodeling, and transcription (2, 13).The signal transducer and activator of transcription (STAT) family of transcription factors mediates a multitude of cytokineregulated gene transcription (reviewed in refs. 14 and 15). In response to ligand binding to cell surface receptors, the STATs are activated through tyrosine phosphorylation, form dimers, enter the nucleus, and bind to specific DNA sequences for transcription activation. The transcriptional activity of STATs are mediated by the transcription activation domain (TAD) located in the C terminus of the molecule (16). The STAT TADs can function independently of the rest of the STAT molecule, and their activit...

Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.In eukaryotes, DNA replication initiates at specific sites called replication origins. cis-acting sequences called replicators define the positions and regulate the activity of replication origins by promoting the assembly of prereplicative complexes (pre-RCs) during the G 1 phase of the cell cycle. Eukaryotic replicators were first identified in the budding yeast Saccharomyces cerevisiae on the basis of their ability to promote the extrachromosomal maintenance of plasmids (22, 50). The dissection of these autonomously replicating sequence (ARS) elements revealed an essential 11-bp sequence, called the ARS consensus sequence (ACS), that is required for both plasmid and chromosomal replicator activity (reviewed in reference 34). The ACS is the core of the binding site for the highly conserved six-subunit origin recognition complex (ORC), which recruits and assembles the other components of the pre-RC (reviewed in reference 1). Like the ORC, the other components of the pre-RC are highly conserved throughout the eukaryotic kingdom.Upon entry into S phase, pre-RCs are activated to initiate replication according to a temporal program whose determinants are poorly understood. The activation of replication origins requires the activity of two kinases, a cyclin-dependent kinase composed of the catalytic subunit encoded by CDC28 and regulatory subunits encoded by CLB5 and CLB6, and the Dbf4-dependent kinase, composed of a catalytic subunit encoded by CDC7 and a regulatory subunit encoded by DBF4. During replication initiation, DNA is unwound at origins and replication fork proteins are assembled to form repl...

Background: IL-6/Stat3 promote breast cancer metastasis through regulation of the fascin gene. Results: In addition to IL-6, TNF-␣ induces binding of a Stat3⅐NFB complex to the fascin promoter to induce transcription. Conclusion: Both NFB and Stat3 are required for cytokine-induced fascin expression and cell migration. Significance: Identification of proteins critical for breast cancer metastasis will reveal drug targets.