title: Interference in transcription of overexpressed genes by promoter-proximal downstream sequences.
abstract: Despite a high sequence homology among four human RNAi-effectors Argonaute proteins and their coding sequences, the efficiency of ectopic overexpression of AGO3 and AGO4 coding sequences in human cells is greatly reduced as compared to AGO1 and AGO2. While investigating this phenomenon, we documented the existence of previously uncharacterized mechanism of gene expression regulation, which is manifested in greatly varying basal transcription levels from the RNApolII promoters depending on the promoter-proximal downstream sequences. Specifically, we show that distinct overexpression of Argonaute coding sequences cannot be explained by mRNA degradation in the cytoplasm or nucleus, and exhibits on transcriptional level. Furthermore, the first 1000-2000 nt located immediately downstream the promoter had the most critical influence on ectopic gene overexpression. The transcription inhibiting effect, associated with those downstream sequences, subsided with increasing distance to the promoter and positively correlated with promoter strength. We hypothesize that the same mechanism, which we named promoter proximal inhibition (PPI), could generally contribute to basal transcription levels of genes, and could be mainly responsible for the essence of difficult-to-express recombinant proteins. Finally, our data reveal that expression of recombinant proteins in human cells can be greatly enhanced by using more permissive promoter adjacent downstream sequences.
authors.affiliation: Molecular Epidemiology C080, German Cancer Research Center, Im Neuenheimer Feld 581, Heidelberg, Germany.|Molecular Biology of Breast Cancer, Department of Gynecology and Obstetrics, Im Neuenheimer Feld 440, Heidelberg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Turchinovich
authors.affiliation: Molecular Epidemiology C080, German Cancer Research Center, Im Neuenheimer Feld 581, Heidelberg, Germany.|Molecular Biology of Breast Cancer, Department of Gynecology and Obstetrics, Im Neuenheimer Feld 440, Heidelberg, Germany.
authors.forename: H M
authors.initials: HM
authors.lastname: Surowy
authors.affiliation: P. Hertsen Moscow Oncology Research Institute, National Center of Medical Radiological Research, 3 Second Botkinsky Lane, Moscow, 125284, Russia.
authors.forename: A G
authors.initials: AG
authors.lastname: Tonevitsky
authors.affiliation: Molecular Epidemiology C080, German Cancer Research Center, Im Neuenheimer Feld 581, Heidelberg, Germany.|Molecular Biology of Breast Cancer, Department of Gynecology and Obstetrics, Im Neuenheimer Feld 440, Heidelberg, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Burwinkel
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000235:genetics*; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D060565:Argonaute Proteins; D012333:RNA, Messenger; D012319:RNA Polymerase II