The transcription factor Myc is induced by mitogenic signals and regulates downstream cellular responses. If overexpressed, Myc promotes malignant transformation. Myc modulates expression of diverse genes in experimental systems, but few are proven direct targets. Here, we present a large-scale screen for genomic Myc-binding sites in live human cells. We used bioinformatics to select consensus DNA elements (CACGTG or E-boxes) situated in the 5 regulatory region of genes and measured Myc binding to those sequences in vivo by quantitative chromatin immunoprecipitation. Strikingly, most promoter-associated E-boxes showed selective recovery with Myc, unlike non-E-box promoters or E-boxes in bulk genomic DNA. Promoter E-boxes were distributed in two groups bound by Myc at distinct frequencies. The high-affinity group included an estimated 11% of all cellular loci, was highly conserved among different cells, and was bound independently of Myc expression levels. Overexpressed Myc associated at increased frequency with low-affinity targets and, at extreme levels, also with other sequences, suggesting that some binding was not sequence-specific. The strongest DNA-sequence parameter defining high-affinity targets was the location of E-boxes within CpG islands, correlating with an open, preacetylated state of chromatin. Myc further enhanced histone acetylation, with or without accompanying induction of mRNA expression. Our findings point to a high regulatory and biological diversity among Myc-target genes.[Keywords: Human; genome; chromatin; transcription factor; Myc] Supplemental material is available at http://www.genesdev.org.

The Myc protein binds DNA and activates transcription by mechanisms that are still unclear. We used chromatin immunoprecipitation (ChIP) to evaluate Myc-dependent changes in histone acetylation at seven target loci. Upon serum stimulation of Rat1 fibroblasts, Myc associated with chromatin, histone H4 became locally hyperacetylated, and gene expression was induced. These responses were lost or severely impaired in Myc-deficient cells, but were restored by adenoviral delivery of Myc simultaneous with mitogenic stimulation. When targeted to chromatin in the absence of mitogens, Myc directly induced H4 acetylation. In addition, Myc recruited TRRAP to chromatin, consistent with a role for this cofactor in histone acetylation. Finally, unlike serum, Myc alone was very inefficient in inducing expression of most target genes. Myc therefore governs a step, most likely H4 acetylation, that is required but not sufficient for transcriptional activation. We propose that Myc acts as a permissive factor, allowing additional signals to activate target promoters.

The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. Here, we show that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC-induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC-target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals.

SUMMARY The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during embryonic development, tissue regeneration and carcinoma progression. Yet many mechanisms by which ERK exerts this control remain elusive. Here, we demonstrate that the ERK-activated kinase RSK is necessary to induce motility and invasive capacities in non-transformed epithelial cells and carcinoma cells. RSK is moreover sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of potent pro-motile/invasive gene program by FRA1-dependent and independent mechanisms. Strikingly, the program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a general mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanates multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties.

ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of ␣L␤ 2 integrins at the plasma membrane of lymphocytes.

E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G 1 following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.

The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. Here we demonstrate that MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts led to rapid cellular senescence that could not be suppressed by hTERT expression. We propose that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence.Supplemental material is available at http://parma.fhcrc.org/ CGrandori. Alterations in c-myc oncogene expression have been implicated in the pathogenesis of several human cancers, including Burkitt and diffuse large B-cell lymphomas, breast and prostate cancer, colon cancer, melanoma, and multiple myeloma (Nesbit et al. 1999). The MYC oncoprotein is a basic helix-loop-helix-leucine zipper (bHLHZIP) transcription factor that through dimerization with MAX protein binds to specific DNA elements ("E boxes") and modulates transcription of a wide variety of genes (for review, see Dang 1999;Grandori et al. 2000;Oster et al. 2002). The proteins encoded by MYC transcriptional target genes appear to regulate cell-cycle progression and cell growth while sensitizing cells to apoptotic stimuli (Evan et al. 1992). MYC may also be able to promote tumorigenesis by up-regulating the expression of genes such as hTERT that play a role in cellular immortalization or the escape from senescence (Wang et al. 1998a;Greenberg et al. 1999;Wu et al. 1999). We reasoned that MYC might modulate the expression of other genes that control cellular senescence, and thus determined whether the gene encoding the Werner syndrome RecQ helicase protein is a MYC transcriptional target.Werner syndrome (WRN) is an uncommon, autosomal recessive genetic instability syndrome that results from loss-of-function mutations in the chromosome 8p12-p11.2 WRN gene (Yu et al. 1996). The WRN phenotype resembles premature aging, and includes genomic instability, an elevated risk of malignancy, and accelerated cellular senescence. Genetic instability following loss of the 162-kD WRN RecQ helicase protein reflects the physiologic role of WRN in mitotic recombination and repair (Brosh and Bohr 2002;Saintigny et al. 2002). Conversely, the elevated levels of WRN observed in immortalized and human tumor cell lines may help insure continuous cell proliferation (Shiratori et al. 1999). In order to delineate potential interactions between MYC and WRN in tumorigenesis, we determined whether WRN expression is modulated by MYC, and monitored cellular responses to MYC overexpression in the absence of WRN. The results indicated that WRN expression appears to be required to avoid cellular senescence upon MYC up-regulation in hTERT-immortalized fibr...