Original information
Original link:
https://onlinelibrary.wiley.com/doi/full/10.1111/tpj.16781
Outline of Research
In a recent study published in The Plant Journal, a research team led by Professor Severin Sasso of the University of Leipzig in Germany has conducted an in-depth identification and characterization of cis-activated regulatory elements (CREs) in the histone gene of Chlamydomonas reinhardtii. This study not only reveals new mechanisms of gene expression regulation and deepens our understanding of the functions of enhancers and CREs in eukaryotes, but also provides key genetic tools and molecular biological foundations for the application of Chlamydomonas reinhardtii in biotechnology, such as bioenergy production, environmental remediation, and crop improvement.
Background:
Cis-Regulatory Module (CRM) is a specific DNA sequence in the genome that is responsible for regulating the expression pattern of genes. CRMs are composed of a variety of cis-regulatory elements (CRE), such as promoters, enhancers, silencers, and insulators. By binding to trans agents such as transcription factors, CRMs can enhance or inhibit gene transcription, thereby regulating the intensity, temporal and spatial distribution of gene expression. CRMs play a crucial role in the development of organisms, cell type-specific expression, and response to environmental changes, and are key components of gene expression regulatory networks in organisms.
Key results
1. Identification of conserved CREs in histones
In the histone genes of Chlamydomonas reinhardtii, the researchers identified four conserved cis-activating elements (CREs), among which EH3/H4 and EH2A/H2B were conserved in the interhistone region of Chlamydomonas reinhardtii, Eupstr was highly enriched at about 65 bp upstream of the transcriptional start site (TSS) of the highly expressed nuclear genes, and the Ehist cons element was ubiquitous in angiosperms and Chlamydomonas reinhardtii histone gene regions. A highly conserved 8-base (called Oct motif) sequence (Figure 1).
图1 EH3/H4、EH2A/H2B、Eupstr和Ehist cons元件的相对位置和它们在基因组中的保守性。
2. Construction and activity testing of reporter vectors
The researchers constructed a reporter vector containing the 4X CRE, promoter, and mCerulean3 fluorescent protein genes and integrated them into the nuclear genome of Chlamydomonas reinhardtii to quantify CRE activation activity by flow cytometry (Figure 2). In the activity evaluation of the four candidate activating CRE, it was found that the two elements, EH3/H4 and EH2A/H2B, did not show upregulation of reporter gene expression. Eupstr significantly enhanced the expression of mCerulean3 fluorescent protein, and its enhancement was comparable to that of the strong promoter PHSP70A-RBCS2 (Figure 3).
Figure 2: Experimental flowchart: Vectors containing CRE, promoter, and mCerulean3 fluorescent reporter were transferred to the nuclear genome of Chlamydomonas reinhardtii by electroporation, and then the expression of the reporter gene was quantified by flow cytometry.
Figure 3: Activity assessment of four candidate activating CREs (EH3/H4, EH2A/H2B, Eupstr, and Ehist cons). The mCerulean3 fluorescence intensity, as measured by flow cytometry, can be used to compare whether these CREs are able to enhance the activity of the PRBCS2 promoter.
3. Explore the factors that affect the activation effect of Eupstr
In this paper, the researchers further investigated the functional properties of Eupstr, a cis-activated regulatory element (CRE). 1. The relationship between activity and quantity of Eupstr was explored, and when 8X Eupstr was used to bind to the RBCS2 promoter, the expression level of mCerulean3 fluorescent protein decreased to a level comparable to that of the RBCS2 promoter alone, and increasing the number of CRE to 8 copies did not enhance its activation (Figure 4b). 2. Binding of Eupstr to the strong promoter PHSP70A-RBCS2 does not enhance its activating activity (Figure 4c). 3. The activation effect of Eupstr is position-dependent, but direction-independent. When Eupstr is located upstream of the RBCS2 promoter, gene expression is significantly increased, whereas when Eupstr is located downstream, this activation effect disappears, and the change in direction of Eupstr does not affect its activating activity (Figure 5). 4. When testing the distance dependence of Eupstr to the promoter, by inserting a 1.5-kb interval between the Eupstr and the promoter, it was found that Eupstr failed to activate the RBCS2 promoter at this distance, suggesting that the activation of Eupstr has a certain distance limitation (Figure 7b), whereas Ehist cons can improve gene expression even at a distance of 1.5-kb (Figure 7c). These findings provide important insights into the role of Eupstr in the regulation of gene expression and provide experimental evidence for the future application of Eupstr as a genetic tool in Chlamydomonas reinhardtii and other possible biological systems.
Figure 4 The effect of 8X Eupstr and the combination with PHSP70A-RBCS2 was evaluated.
Fig. 5 The position and direction dependence of Eupstr was evaluated.
Fig.7 The distance dependence of Eupstr and Ehist cons was evaluated.
4. The potential of cis-activated regulatory elements in biotechnology applications
The application of activated CREs in the field of biotechnology, especially in genetic engineering, has been widely explored and utilized in angiosperms, but relatively few applications in Chlamydomonas reinhardtii, which may be due to the lack of data on the long-term stability of gene expression activated by CREs. To test whether activating CREs can support long-term gene expression, the researchers transformed a reporter vector containing Eupstr bound to the RBCS2 promoter into Chlamydomonas reinhardtii. Flow cytometry analysis of the transformed cells showed that the proportion of cells with high expression of mCerulean3 decreased after screening and passaging, indicating that the expression of overexpressed nuclear genes in Chlamydomonas reinhardtii was moderately stable.
Fig.8 Stability of Eupstr-activated mCerulean3 expression.
The researchers further validated the activation of Eupstr in a patented strain, UVM11-CW, where the PRBCS2 promoter was able to effectively drive high-level gene expression even in the absence of cis-activating elements (CRE). The presence of Eupstr further enhances the expression of reporter genes, which further confirms the effectiveness of activated CREs in enhancing gene expression. Overall, the two activating CREs, Eupstr and Ehist cons, have been shown to activate gene expression in the nuclear genome of Chlamydomonas reinhardtii. These specific CRE-promoter combinations not only provide new strategies for achieving overexpression of target genes, but also open up new possibilities for other biotechnology applications, such as synthetic biology and genetic engineering.
Fig.9 Gene upregulation of Eupstr in Chlamydomonas reinhardtii UVM11-CW strain.
conclusion
In this paper, we studied the cis-activated regulatory elements (CREs) of histone genes in Chlamydomonas reinhardtii in depth, and by constructing a reporter gene system and using flow cytometry, two CREs, namely Eupstr and Ehist cons, were found to have significantly upregulated gene expression. Eupstr is a direction-independent CRE capable of activating RBCS2 and β2-tubulin promoters, while Ehist cons shows binding specificity to specific promoters. In addition, the study explores the location, orientation, and distance dependence of these CREs, as well as their potential for application in biotechnology. These findings not only expand our understanding of the regulatory mechanisms of Chlamydomonas reinhardtii gene expression, but also provide new tools for genetic engineering and synthetic biology research using these cis-regulatory elements.
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