Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5 cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen-and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogeninduced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.Mitogens stimulate protein and RNA synthesis (56, 65). The increase in protein synthesis is partly due to increased initiation on preexisting mRNAs, with the result that those mRNAs are recruited into larger polysomes. In addition to an increase in basal translation, specific mRNAs are preferentially upregulated, suggesting that mitogenic signal transduction pathways impinge on the components of the translation machinery that interact with the mRNA. mRNAs are brought to the ribosome by eukaryotic initiation factor (eIF4F) (for reviews, see references 58, 60, and 61). eIF4F is a multiprotein complex formed from 25-, 46-and 220-kDa subunits, called eIF4E, eIF4A, and eIF4G, respectively. eIF4E, also known as cap-binding protein, is responsible for binding the 5Ј-terminal 7-methyl-GTP (m 7 GTP) cap found on all eukaryotic mRNAs. eIF4A is a subunit of an RNA helicase that seems to unwind secondary structure in the mRNA. eIF4G is the scaffolding subunit, to which the other subunits bind. It also has a binding site for eIF3, which links the eIF4F-mRNA complex to the 40S ribosomal subunit. In yeast, eIF4G has an additional functional region, to which the poly(A)-binding protein and the 3Ј end of the mRNA bind (64). Besides serving as a passive scaffold, eIF4G plays a regulatory role, stimulating the binding of capped mRNA to eIF4E (24). Thus, the eIF4F complex promotes interactions between the 5Ј end of the mRNA, the ribosome, and an RNA helicase.As the main mRNA-binding component of the translation machinery, the eIF4F complex has the potential to distinguish between mRNAs for differential translat...

Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.

We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.

SUMMARY Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane bound compartment—the inclusion--and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia’s intracellular lifecycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, resulting in SNX5/6 relocalization to the inclusion membrane and enhanced inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.

We used a combination of genome‐wide and promoter‐specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here, we show that p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression.Supplemental material is available at http://www.genesdev.org. Studies of transcriptional regulation at individual promoters have led to the general model that ordered recruitment of a combination of factors achieves gene-specific transcription (Cosma 2002). Global genomic analysis extends this model to show how complex transcriptional regulatory networks can emerge from the combinatorial regulation of individual genes and has identified classes of simple regulatory motifs, such as feed-forward loops and regulatory cascades ( Fig. 1A) (Lee et al. 2002;Milo et al. 2002;Shen-Orr et al. 2002). A current opportunity for developmental biology is to use these two approaches, promoter-specific molecular biology and systems network biology, to reveal the molecular events that temporally pattern multistage gene expression programs during development and cell differentiation.The differentiation of skeletal muscle is a powerful system for studying the molecular regulation of a multistaged program of cell differentiation. Vertebrate myogenesis is regulated by the bHLH transcription factor MyoD, and its paralogs Myogenin, Myf-5, and MRF4. These act by heterodimerizing with E-proteins and binding CAnnTG recognition sites (Blackwell and Weintraub 1990). Genetic experiments have shown that MyoD or Myf-5 act as lineage-determination factors and Myogenin mediates terminal differentiation (for review, see Arnold and Braun 1996). When expressed in nonmuscle cell types in vitro, each of these factors is sufficient to drive differentiation into skeletal muscle and allows the process to be studied in molecular detail (Weintraub et al. 1989;Choi et al. 1990).Studies of myogenesis have revealed a predictable temporal pattern of gene expression both in vivo and in vitro (Lin et al. 1994;Zhao et al. 2002). In our previous study, we characterized the timing of gene expression associated with muscle differentiation in a model system consisting of mouse embryonic fibroblasts (MEFs) expressing an inducible MyoD-Estrogen Receptor fusion protein (MyoD-ER), which allows synchronized skeletal muscle differentiation (Hollenberg et al. 1993;Bergstrom et al. 2002). Microarray analysis demonstrated that MyoD activity altered the expression of ∼5% of a...

Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis-the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.