Using genome-wide analysis of transcription factor occupancy, we investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. We found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed our predictions. We showed that NRF1, a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. Our studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors.
T cell development and selection require the fully mature and diverse epithelial microenvironment of the thymus. Acquisition of these characteristics is dependent on expression of the forkhead (also known as winged-helix) transcription factor FoxN1, as a lack of functional FoxN1 results in aberrant epithelial morphogenesis and an inability to attract lymphoid precursors to the thymus primordium. However, the transcriptional control of Foxn1 expression has not been elucidated. Here we report that secreted Wnt glycoproteins, expressed by thymic epithelial cells and thymocytes, regulate epithelial Foxn1 expression in both autocrine and paracrine fashions. Wnt molecules therefore provide regulatory signals critical for thymic function.
Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycledependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.E2F/retinoblastoma tumor suppressor (pRB) complexes play a critical role in the regulation of cell cycle progression. There are at least eight E2F transcription factors expressed in mammals (reviewed in references 3 and 29). pRB and related family members regulate the transcription of E2F-responsive genes by interacting with the E2Fs. Three pRB family members, pRB, p130, and p107 (termed pocket proteins), are expressed in mammalian cells. pRB family members in the hypophosphorylated state bind to E2Fs and inhibit transcription. This inhibition is relieved when the pocket proteins are released from E2F complexes following their phosphorylation by cyclin/CDK activity (15). There are functional and structural differences within the E2F family. E2F-1, E2F-2, and E2F-3 are transcriptional activators and interact with pRB. E2F-4 and E2F-5 are transcriptional repressors and preferentially bind p130 and p107 (17). Although E2F-6 apparently does not have a pocket protein interaction domain, it interacts with Polycomb proteins and thus represses transcription (7,18,32,45). The recently identified E2F-7 and E2F-8 are also believed to repress specific promoters (14, 29).Although p107, p130, and pRB are closely related members of the same family, they have different affinities for E2F family members, and they exhibit distinct temporal regulation during the cell cycle. While E2F4/p130 complexes are the most abundant in quiescent cells, E2F4/p107 and E2F4/pRB complexes accumulate in G 1 cells (31). In addition, while p130 and p107 are bound to a number of promoters in asynchronously growing cells, only p...
Since the extracellular matrix (ECM) can promote platelet-derived growth factor (PDGF)-dependent responses, we hypothesized that the ECM mediates this effect by preventing the PDGF  receptor (PDGFR) from associating with the negative regulator, RasGAP (the GTPase-activating protein of Ras). We found that binding of RasGAP to the wild-type PDGFR was decreased; the activation of Ras and Erk was enhanced, and [3 H]thymidine uptake was better in cells cultured on fibronectin than in cells cultured on polylysine. To investigate the mechanism by which culturing cells on fibronectin diminished the recruitment of RasGAP to the PDGFR, we focused on SHP-2 since it dephosphorylates the PDGFR at the phosphotyrosine required for binding of RasGAP. Culturing cells on fibronectin increased the amount of SHP-2 that associated with the PDGFR. Furthermore, cells expressing receptor mutants that failed to associate with SHP-2 were insensitive to fibronectin. The ECM enhances PDGF-dependent responses by increasing the association of SHP-2 with the PDGFR, which in turn decreases the time that Ras-GAP interacts with the receptor. Thus, fibronectin changes PDGF-dependent signaling and biological responses by altering the signal relay enzymes that are recruited to the receptor.
Gel shift analysis reveals [Lagunavicius, A., & Siksnys, V. (1997) Biochemistry 36 (preceding paper in this issue)] that at pH 8.3 in the absence of Mg2+, MunI restriction endonuclease exhibits little DNA binding specificity, as compared with the D83A and E98A mutants of MunI. This suggests that charged carboxylate residue(s) influence the DNA binding specificity of MunI. In our efforts to establish the determinants of MunI binding specificity, we investigated the possible role of the ionic milieu, and we found that lowering pH or elevating Ca2+ levels per se induces specific DNA recognition by WT MunI. In contrast to the binding experiments at pH 8.3, gel shift analysis at pH 6.5 indicated tight sequence-specific binding of WT MunI in the absence of Mg2+, suggesting that protonation of active site carboxylate residue(s) which manifest anomalously high pKa value(s) control binding specificity. Interestingly, Ca2+ ion concentrations, which did not support DNA cleavage by MunI also induced DNA binding specificity in WT MunI at pH 8.3. To explore possible structural changes upon DNA binding, we then used a limited proteolysis technique. Trypsin cleavage of MunI-DNA complexes indicated that in the presence of cognate DNA the MunI restriction endonuclease became resistant to proteolytic cleavage, suggesting that binding of specific DNA induced a structural change. CD measurements confirmed this observation, suggesting minor secondary structural differences between complexes of MunI with cognate and noncognate DNA. These results therefore suggest that binding of MunI to its recognition sequence triggers a conformational transition that correctly juxtaposes active site carboxylate residues, which then chelate Mg2+ ions. In the absence of Mg2+ ions, at pH 8.3, conditions in which carboxylate groups would be expected to be completely ionized, electrostatic repulsion between charged carboxylates and phosphate oxygens is enhanced such as to interfere with specific DNA binding. Elimination of such repulsive constraints by replacement of carboxylate residues, by lowering pH, or by metal ion binding, then promotes MunI binding specificity.
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