ABSTRACT
BACKGROUND: Despite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110delta catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy. PRINCIPAL FINDINGS: We show that p110delta expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110delta gene transcription appears to be constitutive and not subject to acute stimulation. 5'RACE experiments revealed that p110delta mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110delta transcripts are higher in leukocytes compared to non-leukocytes, with the p110delta transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110delta gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a. CONCLUSION: This study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110delta.
Subject(s)
Gene Expression Regulation, Enzymologic , Isoenzymes , Phosphatidylinositol 3-Kinases , 5' Untranslated Regions/genetics , Animals , Base Sequence , Binding Sites , Cell Line , Class I Phosphatidylinositol 3-Kinases , DNA Methylation , Exons , Genes, Reporter , Histones/metabolism , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Leukocytes/physiology , Mice , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Promoter Regions, Genetic , RNA Stability , Sequence Analysis, DNA , Signal Transduction/physiology , Transcription Initiation SiteABSTRACT
DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.
Subject(s)
Dual Specificity Phosphatase 6/metabolism , Fibroblast Growth Factors/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-ets/metabolism , Signal Transduction/physiology , Animals , Base Sequence , Binding Sites , Cell Line , Dual Specificity Phosphatase 6/genetics , Enzyme Activation , Feedback, Physiological/physiology , Gene Expression Regulation , Humans , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-ets/genetics , Sequence Alignment , TransgenesABSTRACT
BACKGROUND: Genome projects have provided a vast amount of sequence information. Sequence comparison between species helps to establish functional catalogues within organisms and to study how they are maintained and modified across phylogenetic groups during evolution. Microarray studies allow us to determine groups of genes with similar temporal regulation and perhaps also common regulatory upstream regions for binding of transcription factors. The integration of sequence and expression data is expected to refine our current annotations and provide some insight into the evolution of gene regulation across organisms. RESULTS: We have investigated how well the protein subcellular localization and functional categories established from clustering of orthologous genes agree with gene-expression data in Saccharomyces cerevisiae. An increase in the resolution of biologically meaningful classes is observed upon the combination of experiments under different conditions. The functional categories deduced by sequence comparison approaches are, in general, preserved at the level of expression and can sometimes interact into larger co-regulated networks, such as the protein translation process. Differences and similarities in the expression between cytoplasmic-mitochondrial and interspecies translation machineries complement evolutionary information from sequence similarity. CONCLUSIONS: Combination of several microarray experiments is a powerful tool for the identification of upstream regulatory motifs of yeast genes involved in protein synthesis. Comparison of these yeast co-regulated genes against the archaeal and bacterial operons indicates that the components of the protein translation process are conserved across organisms at the expression level with minor specific adaptations.
