Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme.
The expression of the transcription factor DeltaLf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with antiproliferative effects and we recently showed that the Skp1 gene promoter was a target of DeltaLf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of DeltaLf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a DeltaLf-M4 mutant corresponding to a constitutively active DeltaLf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of DeltaLf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of DeltaLf- and DeltaLf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(DcpS) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that DeltaLf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of DeltaLf transcriptional activity.
Mariller
Christophe
C
Université des Sciences et Technologies de Lille, CNRS, UMR 8576, Unité de Glycobiologie Structurale et Fonctionnelle, F-59655 Villeneuve d'Ascq cedex, France. christophe.mariller@univ-lille1.fr
Hardivillé
Stephan
S
Hoedt
Esthelle
E
Benaïssa
Monique
M
Mazurier
Joël
J
Pierce
Annick
A
D015153:Blotting, Western; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007781:Lactoferrin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D012333:RNA, Messenger; D044786:S-Phase Kinase-Associated Proteins; D004722:Endoribonucleases; C485332:DcpS protein, human; D007781:Lactoferrin