Article

Identification of transcription factor binding sites upstream of human genes regulated by the phosphatidylinositol 3-kinase and MEK/ERK signaling pathways.

Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 06/2004; 279(19):20167-77. DOI: 10.1074/jbc.M309260200
Source: PubMed

ABSTRACT We have taken an integrated approach in which expression profiling has been combined with the use of small molecule inhibitors and computational analysis of transcription factor binding sites to characterize regulatory sequences of genes that are targets of specific signaling pathways in growth factor-stimulated human cells. T98G cells were stimulated with platelet-derived growth factor (PDGF) and analyzed by DNA microarrays, which identified 74 immediate-early gene transcripts. Cells were then treated with inhibitors to identify subsets of genes that are targets of the phosphatidylinositol 3-kinase (PI3K) and MEK/ERK signaling pathways. Four groups of PDGF-induced genes were defined: independent of PI3K and MEK/ERK signaling, dependent on PI3K signaling, dependent on MEK/ERK signaling, and dependent on both pathways. The upstream regions of all genes in the four groups were scanned using TRANSFAC for putative cis-elements as compared with a background set of non-induced genes. Binding sites for 18 computationally predicted transcription factors were over-represented in the four groups of co-expressed genes compared with the background sequences (p < 0.01). Many of the cis-elements identified were conserved in orthologous mouse genes, and many of the predicted elements and their cognate transcription factors were consistent with previous experimental data. In addition, chromatin immunoprecipitation assays experimentally verified nine predicted SRF binding sites in T98G cells, including a previously unknown SRF site upstream of DUSP5. These results indicate that groups of human genes regulated by discrete intracellular signaling pathways share common cis-regulatory elements.

0 Followers
 · 
98 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: During the initial hours after activation, CD4(+) T cells experience profound changes in gene expression. Co-stimulation via the CD28 receptor is required for efficient activation of naive T cells. However, the transcriptional consequences of CD28 co-stimulation are not completely understood. We performed expression microarray analysis to elucidate the effects of CD28 signals on the transcriptome of activated T cells. We show that the transcription factor DEC1 is highly induced in a CD28-dependent manner upon T cell activation, is involved in essential CD4(+) effector T cell functions, and participates in the transcriptional regulation of several T cell activation pathways, including a large group of CD28-regulated genes. Antigen-specific, DEC1-deficient CD4(+) T cells have cell-intrinsic defects in survival and proliferation. Furthermore, we found that DEC1 is required for the development of experimental autoimmune encephalomyelitis because of its critical role in the production of the proinflammatory cytokines GM-CSF, IFN-γ, and IL-2. Thus, we identify DEC1 as a critical transcriptional mediator in the activation of naive CD4(+) T cells that is required for the development of a T cell-mediated autoimmune disease.
    Journal of Experimental Medicine 07/2013; 210(8). DOI:10.1084/jem.20122387 · 13.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton.
    Genome Research 03/2006; 16(2):197-207. DOI:10.1101/gr.4108706 · 13.85 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Activity based alterations in synaptic connectivity are thought to underlie the processes involved in learning and memory. Measurable changes in neuronal activation by long-term potentiation (LTP) are widely investigated as a possible cellular correlate of this phenomenon, as it can be induced quickly to elicit long-lasting modifications. These long-term changes in the activity of neuronal circuits are sustained by an altered pattern of gene expression and protein synthesis. The inducible transcription factor Zif268 has been implicated in almost all models of neuronal plasticity. Downstream targets of zif268 are widely believed to contribute to the duration and stabilisation of NMDA receptor dependent LTP which, in turn, can be linked to various models of learning and memory. However, these downstream targets are only just starting to receive attention. By utilising a wide range of contemporary neuroscience techniques covering molecular & cell biology approaches, this thesis proposes two known proteins, gephyrin and ubiquilin, as well as a novel gene (urma), as potential downstream targets of Zif268. Both gephyrin and ubiquilin are associated with GABAA receptors at inhibitory synapses. Gephyrin is thought to cluster and anchor GABAA receptors at postsynaptic sites whilst ubiquilin is reported to regulate receptor surface expression. We found that gephyrin mRNA and protein expression levels were downregulated in response to increased levels of zif268 by transient transfection in PC-12 cells and NMDA stimulation in primary cultured cortical neurones. In addition, ubiquilin mRNA and protein levels were also downregulated within the same experimental paradigms, implying that both gephyrin and ubiquilin are downstream transcriptional targets of this plasticity-related gene. A previously reported microarray experiment (James et al. 2005) contained 144 ESTs significantly affected by the transient transfection of Zif268 in PC-12 cells compared to control. Bioinformatic analyses of these tags revealed interesting genomic areas pertaining to little published information. After further investigation, EST AI169020 revealed a novel transcript that was downregulated in response to NMDA treatment of primary cortical neurones. Additional data mining suggests that urma may be a rarely expressed transcription factor. Basal levels of urma mRNA were also decreased in the Zif268 knockout mouse, as were ubiquilin mRNA levels. Zif268 is a regulatory immediate early gene, activating or suppressing downstream targets that play a role in the duration and stabilisation of LTP. These results indicate that gephyrin and ubiquilin are potential mediators of NMDA receptor-dependent plasticity, by modifying inhibitory-signalling. In addition, Zif268 may actively suppress a novel plasticity-related transcription factor, urma. These findings may underlie important processes in learning and memory.

Preview

Download
6 Downloads