Publisher Correction: Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21369-z

Pan-Cancer Analysis of Head-to-Head Gene Pairs in Terms of Transcriptional Activity, Co-expression and Regulation

BackgroundHead-to-Head (H2H) gene pairs are regulated by bidirectional promoters and divergently transcribed from opposite DNA strands with transcription start sites (TSSs) separated within 1 kb. H2H organization is ancient and conserved, and H2H pairs tend to exhibit similar expression patterns. Although some H2H genes have been reported to be associated with disease and cancer, there is a lack of systematic studies on H2H organization in the scenario of cancer development.MethodsHuman H2H gene pairs were identified based on GENCODE hg19 and the functional relevance of H2H pairs was explored through function enrichment and semantic similarity analysis. To investigate the association between H2H organization and carcinogenesis, pan-cancer differential analysis of H2H genes about transcriptional activity, co-expression and transcriptional regulation by transcription factors and enhancers were performed based on data from The Cancer Genome Atlas. Cox proportional hazards regression model and log-rank test were used to determine the prognostic powers of H2H pairs.ResultsIn the present study, we first updated H2H genes from 1,447 to 3,150 pairs, from which the peak group with TSS distance of 1–100 was observed as expected in our previous work. It was found that housekeeping genes, mitochondrial-functional associated genes and cancer genes tend to be organized in H2H arrangement. Pan-cancer analysis indicates that H2H genes are transcriptionally active than random genes in both normal and cancer tissues, but H2H pairs display higher correlation in cancer than in normal. Particularly, housekeeping H2H pairs are differentially correlated much more significantly than non-housekeeping H2H pairs are. Some of differentially correlated H2H pairs were found to be associated with prognosis. The alteration of TF similarity seems to contribute to differential co-expression of H2H pairs during carcinogenesis; meanwhile remote enhancers also at least partly explain the differential co-expression and co-regulation of H2H pairs.ConclusionH2H pairs tend to show much stronger positive expression correlation in cancer than in normal due to differential regulation of bidirectional promoters. The study provides insights into the significance of H2H organization in carcinogenesis and the underlying dysfunctional regulation mechanisms. Those differentially correlated H2H pairs associated with survival have the potential to be prognostic biomarkers and therapeutic targets for cancer.

Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon

Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.

Widespread divergent transcription from prokaryotic promoters

ABSTRACTPromoters are DNA sequences that stimulate the initiation of transcription. In all prokaryotes, promoters are believed to drive transcription in a single direction. Here we show that prokaryotic promoters are frequently bidirectional and drive divergent transcription. Mechanistically, this occurs because key promoter elements have inherent symmetry and often coincide on opposite DNA strands. Reciprocal stimulation between divergent transcription start sites also contributes. Horizontally acquired DNA is enriched for bidirectional promoters suggesting that they represent an early step in prokaryotic promoter evolution.

Bidirectional histone-gene promoters in Aspergillus: characterization and application for multi-gene expression

Background Filamentous fungi are important producers of enzymes and bioactive secondary metabolites and are exploited for industrial purposes. Expression and characterization of biosynthetic pathways requires stable expression of multiple genes in the production host. Fungal promoters are indispensable for the accomplishment of this task, and libraries of promoters that show functionality across diverse fungal species facilitate synthetic biology approaches, pathway expression, and cell-factory construction. Results In this study, we characterized the intergenic region between the genes encoding histones H4.1 and H3, from five phylogenetically diverse species of Aspergillus, as bidirectional promoters (Ph4h3). By expression of the genes encoding fluorescent proteins mRFP1 and mCitrine, we show at the translational and transcriptional level that this region from diverse species is applicable as strong and constitutive bidirectional promoters in Aspergillus nidulans. Bioinformatic analysis showed that the divergent gene orientation of h4.1 and h3 appears maintained among fungi, and that the Ph4h3 display conserved DNA motifs among the investigated 85 Aspergilli. Two of the heterologous Ph4h3s were utilized for single-locus expression of four genes from the putative malformin producing pathway from Aspergillus brasiliensis in A. nidulans. Strikingly, heterologous expression of mlfA encoding the non-ribosomal peptide synthetase is sufficient for biosynthesis of malformins in A. nidulans, which indicates an iterative use of one adenylation domain in the enzyme. However, this resulted in highly stressed colonies, which was reverted to a healthy phenotype by co-expressing the residual four genes from the putative biosynthetic gene cluster. Conclusions Our study has documented that Ph4h3 is a strong constitutive bidirectional promoter and a valuable new addition to the genetic toolbox of at least the genus Aspergillus.