The Drosophila nuclear factor DREF positively regulates the expression of the mitochondrial transcription termination factor DmTTF.
The DREF [DRE (DNA replication-related element)-binding factor], which regulates the transcription of a group of cell proliferation-related genes in Drosophila, also controls the expression of three genes involved in mtDNA (mitochondrial DNA) replication and maintenance. In the present study, by in silico analysis, we have identified DREs in the promoter region of a gene participating in mtDNA transcription, the DmTTF (Drosophila mitochondrial transcription termination factor). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DREs control DmTTF transcription; moreover, gel-shift and ChIP (chromatin immunoprecipitation) assays demonstrated that the analysed DRE sites interact with DREF in vitro and in vivo. Accordingly, DREF knock-down in S2 cells by RNAi (RNA interference) induced a considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the other hand, mtRNApol (mitochondrial RNA polymerase) lacks DREs in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mtRNApol was efficiently transcribed from stages 11-12. Territories where transcription occurred mostly were the gut and Malpighi tubes for DmTTF, and the gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiple roles in the mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.
Fernández-Moreno
Miguel A
MA
Departamento de Bioquímica, Instituto de Investigaciones Biomedicas 'Alberto Sols' CSIC-UAM, Universidad Autónoma de Madrid, c/Arzobispo Morcillo 4, 28029 Madrid, Spain. miguel.fernandez@uam.es
Bruni
Francesco
F
Adán
Cristina
C
Sierra
Rosana Hernández
RH
Polosa
Paola Loguercio
PL
Cantatore
Palmiro
P
Garesse
Rafael
R
Roberti
Marina
M
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004331:Drosophila melanogaster / Q000235:genetics*; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D024101:Mitochondrial Proteins / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D012318:RNA Polymerase I / Q000502:physiology; D020218:Response Elements; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C099494:Dref protein, Drosophila; D029721:Drosophila Proteins; D024101:Mitochondrial Proteins; D014157:Transcription Factors; C501534:mTTF protein, Drosophila; D012318:RNA Polymerase I