Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes

Protein complexes that bind to ‘GAGA’ DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p < 1 × 10^− 8). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution.     

A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.     

Hepatocyte-specific gene expression from integrated lentiviral vectors

BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.     

5′ Regulatory region of ubiquitin 2 gene from Porteresia coarctata makes efficient promoters for transgene expression in monocots and dicots

Key message

Porteresia ubiquitin 5′ regulatory region drives transgene expression in monocots and dicots.

Abstract

Ubiquitin promoters are promising candidates for constitutive transgene expression in plants. In this study, we isolated and characterized a novel 5′ regulatory sequence of a ubiquitin gene from Porteresia coarctata, a stress-tolerant wild grass species. Through functional analysis in heterologous plant systems, we have demonstrated that full length (Port Ubi2.3) or truncated sequence (PD2) of the isolated regulatory fragment can drive constitutive expression of GUS in monocots and/or dicots. In silico analysis of Port Ubi2.3 has revealed the presence of a 640 bp core promoter region followed by two exons and two introns with numerous putative cis-acting sites scattered throughout the regulatory region. Transformation and expression studies of six different deletion constructs in rice, tobacco and sugarcane revealed that the proximal intron has an enhancing effect on the activity of the core promoter in both monocots and dicots, whereas, Port Ubi2.3 was able to render strong expression only in monocots. This regulatory sequence is quite distinct from the other reported ubiquitin promoters in structure and performs better in monocots compared to other commonly used promoters—maize Ubi1 and Cauliflower Mosaic Virus 35S.     

Cis-regulatory elements used to control gene expression in plants

Plant biotechnology is a dynamically developing science, which comprises many fields of knowledge. Novel plant genetic engineering findings highly influence the improvement of industrial production. These findings mostly concern cis-regulatory elements, which are sequences controlling gene expression at all developmental stages. They comprise of promoters, enhancers, insulators and silencers, which are used to construct synthetic expression cassettes. Examples of most important cis-regulatory elements are reviewed in the present paper. Variability among core promoters content and distal promoter regions impedes evaluation of interactions between them during the artificial promoters construction. Synthetic promoters and artificial expression cassettes trigger a significant increase in gene expression level, better properties and quality of a product. Accumulating knowledge about gene promoters, cis sequences and their cooperating factors allows uniform expression systems and highly predictable results.     

Construction of Stress Responsive Synthetic Promoters and Analysis of Their Activity in Transgenic Arabidopsis thaliana

To obtain strong inducible promoters to drive abiotic stress-inducible transgene expression with minimal negative effects, we constructed three artificial synthetic promoters (EKCM, EKCRM, and ECCRM) comprising multiple cis-acting stress-response elements. Each promoter was fused independently to the β-glucuronidase (GUS) reporter gene, and GUS expression was analyzed in stable expression systems in Arabidopsis thaliana. T2 transgenic progenies showed integration of the promoter-GUS construct in their genome. RT-PCR assays and histochemical staining analysis showed that GUS expression driven by each promoter increased under desiccation, cold, and high salt conditions. The activity of synthetic promoters, assessed by fluorometric quantitative analysis of GUS enzyme activity, was significantly higher than that of the rd29A promoter under various stress treatments. The most powerful promoter, EKCM, allowed about 1.29-fold in GUS activity relative to the rd29A promoter, on average, under dehydration conditions. All three synthetic promoters could drive stress-inducible GUS expression in different organs of transgenic Arabidopsis. These synthetic promoters represent valuable tools for improving the stress tolerance of crops.