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The effect of autonomously replicating sequences on gene expression in saccharomyces cerevisiae
Biochemical Engineering Journal 149 (2019) 107250
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
The effect of autonomously replicating sequences on gene expression in saccharomyces cerevisiae
T
Xiao-Le Wua,b, Yan-Hui Bia,b, Feng Gaoa,c, Ze-Xiong Xiea,b, Xia Lia,b, Xiao Zhoua,b, De-Jun Mad, ⁎ Bing-Zhi Lia,b, , Ying-Jin Yuana,b a
Frontier Science Center of Synthetic Biology (MOE), Key Laboratory of Systems Bioengineering (MOE), Tianjin University, Tianjin, 300072, China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China c Department of Physics, Tianjin University, Tianjin, 300072, China d State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China b
H I GH L IG H T S
replicating sequence (ARS) activity affected adjacent gene expression. • Autonomously active ARSs promoted gene expression in a non-synergistic manner. • Strongly of strongly active ARSs decreased adjacent gene expression. • Knockout • Chromosome structure influenced the effects of ARSs on gene expression.
A R T I C LE I N FO
A B S T R A C T
Keywords: Gene expression ARS Chromosome structure Synthetic biology Yeast
Autonomously replicating sequences (ARSs) are essential for genome replication and can regulate gene expression as potential cis-elements; however, the mechanisms underlying this regulation are unclear. In order to understand these mechanisms, we first replaced genes adjacent to ARSs on chromosome III of Saccharomyces cerevisiae with an expression cassette containing the gene encoding red fluorescence protein (RFP) and verified that the relative fluorescence intensity of RFP was positively correlated with the activities of the adjacent ARSs. Then, to investigate the effect of ARSs on gene expression, we designed ARS-insertion and -deletion variants, finding that the insertion of a strongly active ARS adjacent to a weakly active ARS enhanced adjacent gene expression, whereas the deletion of a strongly active ARS reduced the expression of the flanking RFP; however, insertion of multiple strong ARSs did not show a synergistic effect on gene expression. Further analysis suggested that ARSs located in compact chromosomal regions exerted a stronger effect on gene expression than those in relaxed chromosomal regions. These results demonstrated that ARSs regulated S. cerevisiae gene expression by interacting with the chromosome structure and provide a potential strategy for regulating gene expression in synthetic biology and metabolic engineering studies using S. cerevisiae.
1. Introduction Unlike prokaryotes, the initiation of DNA replication usually occurs at discrete chromosomal loci in yeast and higher eukaryotes [1–3]. In the eukaryotic cell cycle, replication is activated at different time points [4]; previous studies identified autonomously replicating sequence
(ARS) elements in budding yeast Saccharomyces cerevisiae as key elements involved in initiating DNA replication and maintaining genome stability [5–7]. Further studies indicated that ARS initiation time and efficiency varied in the yeast chromosome [8–12]. A previous study examined the ARS activity by analyzing the replication origin efficiency of DNA fragments carrying the ARS elements [13]. The different shapes
Abbreviations: 2D, two dimensional; 3D, three dimensional; ARS, autonomously replicating sequence; gRNA, genomic RNA; MAR, matrix attachment region; PCR, polymerase chain reaction; rARS, ribosomal ARS; RFP, red fluorescent protein; YKO, yeast knockout ⁎ Corresponding author at: Frontier Science Center of Synthetic Biology (MOE), Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China. E-mail address: [email protected] (B.-Z. Li). https://doi.org/10.1016/j.bej.2019.107250 Received 21 February 2019; Received in revised form 27 May 2019; Accepted 28 May 2019 Available online 29 May 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
Biochemical Engineering Journal 149 (2019) 107250
X.-L. Wu, et al.
created by adding 20 mg/L uracil to SC-Leu-Ura media and SC-Ura media was created by adding 20 mg/L leucine to SC-Leu-Ura media [25]. Amino acids were purchased from Genview (Beijing, China).
of replication intermediates could be separated by two-dimensional (2D) electrophoresis. The "bubble-shaped" replication intermediates represented an active origin and "Y-shaped" intermediates reflected a passive replication by a fork from an origin external to the fragment [14]. The replication origin activity was revealed by the occurrence of bubble shape replication intermediates, which varied from 90% to < 10% [9,10]. Recently, increasing evidence has shown that ARSs are associated with adjacent gene expression [15]. The integration of heterogeneous genes at different loci in the yeast genome resulted in significant differences in gene-expression levels [16–18], suggesting that ARSs might be a factor affecting gene expression. Upon subsequent random insertion of the heterogeneous genes at 20 loci in the yeast genome, the highest transcriptional activity was observed when the reported gene was inserted within 400 bp of the closest ARS [19]. Matrix attachment regions (MARs) in higher eukaryotes, such as Drosophila [20] and fish [21], exhibit similar functions involving DNA replication as ARSs in the yeast, with one study demonstrating that the MARs integrated within an S. cerevisiae plasmid affected the expression of enhanced green fluorescent protein [21]. Additionally, the replication activity of an ARS might be a prerequisite for regulating gene expression. A study of a ribosomal ARS (rARS) demonstrated that ribosomal RNA genes flanking activated rARSs were preferentially active in S. cerevisiae [15], and our previous study indicated that only reporter genes close to strongly active ARSs exhibited high expression levels [22]. Our previous study investigated ARS functions on gene expression in situ [22]. Therefore, in the present study, we ectopically integrated strongly active ARSs on chromosome III of S. cerevisiae in order to examine their effects on the expression of nearby genes. Additionally, we knocked out five strongly active ARSs and explored the effects on the expression of adjacent genes, with subsequent analyses of ARS-specific effects on gene expression in both the chromosome and a plasmid.
2.2. Construction of expression cassettes We used RFP as the reporter gene with the pURA3 promoter cloned from an existing plasmid maintained in our lab. The homology arms (kanMX-L and kanMX-R) were cloned from the genome of the YKO collection, and ARS305 was cloned from the genome of BY4742. DNA fragments were integrated into the pUC19 plasmid and cloned for the subsequent transformation procedures. 2.3. Construction of guide RNA expression plasmids A genomic RNA (gRNA)-expression plasmid targeting a specific genomic locus was constructed according to a previous method [26,27]. First, the pRS426-SNR52p-gRNA-SUP4t plasmid (stored in our lab) was digested by NotI (New England Biolabs, Ipswich, MA, USA) and purified by agarose gel electrophoresis. Second, the oligonucleotide primers were annealed for gRNA-expression plasmid construction. The primers included a 20-bp gRNA fragment and 40-bp overlapping sequence, with pRS426-SNR52p-gRNA-SUP4t located at the cleavage site. Third, the 60-bp fragment and linear plasmid were connected through Gibson assembly (New England Biolabs, Ipswich, MA, USA) and transformed into Escherichia coli [28]. 2.4. CRISPR/Cas9-based transformation A CRISPR/Cas9-based transformation strategy was used to substitute the kanMX cassettes and ARS, as described previously [29]. First, the Cas9-expression plasmid constructed from plasmid pRS415 was transformed into strains, followed by incubation on SC-Leu plates at 30 °C for 2 days [26]. Polymerase chain reaction (PCR) was used to confirm the correct strains. Second, we transformed both the gRNA plasmid and the DNA fragment into the strain, with SC-Leu-Ura medium used as a screening medium. After completion of the transformation, cells were plated and incubated for 2 days at 30 °C, and PCR was used to verify candidate colonies.
2. Materials and methods 2.1. Strains and media Recombinant strains were derived from a yeast knockout (YKO) collection using homologous recombination [23,24] and are listed in Supplementary Table 1. The media included SC-Leu-Ura, SC-Leu, and SC-Ura. SC-Leu-Ura was a synthetic dextrose medium containing 6.7 g/L yeast nitrogen base without amino acids (Solarbio, Beijing, China), 20 g/L glucose, 2 g/L complete supplement mixture (without histidine, leucine, tryptophan, uracil), 20 mg/L histidine, and 20 mg/L tryptophan. SC-Leu media was
2.5. RFP expression in plasmids Plasmids carrying different ARSs were constructed based on the pRS416 plasmid [30]. First, we used PCR to obtain the pRS416 fragment lacking the ARS, followed by cloning of the ARSs in chromosome Fig. 1. Homologous recombination and vector configuration. (A) The RFP gene was integrated into loci adjacent to ARSs on chromosome III by replacing the kanMX gene. The DNA fragment used included RFP with the pURA3 promoter and two homology arms (KanMX-L and KanMX-R). (B) Strongly active ARS305 together with RFP were integrated into loci adjacent to ARSs on chromosome III. The DNA fragment included ARS305, RFP with pURA3, and two homology arms (kanMX-L and kanMX-R). (C) Knockout of strongly active ARS (ARSa) on chromosome III and insertion of RFP at loci adjacent to the deleted ARSs. Two DNA fragments were used: one included the homologous arms on both sides of the ARS, and the other was the same as the fragment used in (A). (D) The ARS fragment in the pRS416 plasmid was replaced with four strongly active or four weakly active ARSs, followed by insertion of RFP. 2
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strongly active ARSs was > 7 (average intensity: 8.56), which was significantly higher than the average relative fluorescence intensity (4.24) associated with RFP inserted adjacent to weakly active ARSs (Fig. 2B).
III from the yeast genome. We then performed Gibson assembly to connect the ARS fragment to the plasmid lacking the ARS (Fig. 1D), followed by transformation of the BY4742 strain with the recombinant plasmids, respectively, with SC-Ura medium used as a screening medium. After transformation, cells were plated and incubated for 2 days at 30 °C, and PCR was preformed used to confirm successful transformation.
3.2. Strongly active ARSs affected gene expression in a dominant rather than additive manner To further investigate the effect of ARS activity on gene expression, the strongly active ARS305 adjacent to RFP was integrated into loci adjacent to other ARSs on chromosome III, including six strongly active and eight weakly active ARSs (Fig. 1B). We found that the expression of RFP inserted near weakly active ARSs, except ARS304 and ARS313, was significantly elevated due to the ARS305 insertion, with the relative fluorescence intensity associated with RFP inserted adjacent to ARS300, ARS301/302/303, ARS320, ARS308, ARS316, and ARS317/318 increasing from 1.79, 3.51, 2.99, 4.71, 5.82, and 3.03 to 4.49, 5.42, 4.87, 6.98, 8.04, and 9.75, respectively (Fig. 3A). However, insertion of ARS305 did not enhance the fluorescence of RFP inserted adjacent to strongly active ARSs, except ARS 315 (from 9.81 to 11.37), suggesting that ARS-related influence on gene expression was not synergetic (Fig. 3B).
2.6. Fluorescence assays Selected strains harboring the reporter gene were grown overnight and resuspended to an absorbance at 600 nm of 0.2, followed by incubating at 30 °C for 4 h. Yeast cells were harvested by centrifugation at 4000 × g for 2 min and resuspended in phosphate-buffered saline. Fluorescence assays were performed individually in cuvettes, and RFP fluorescence was measured using a SpectraMax M2 detector (Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 587 nm and an emission wavelength of 610 nm [31,32]. Relative fluorescence intensity was derived as fluorescence/optical density at 600 nm. 3. Results 3.1. Positive relationship between gene expression and ARS activity
3.3. Knockout of strongly active ARSs decreased gene expression
There are 20 ARSs on chromosome III of S. cerevisiae according to the DNA Replication Origin Database (http://cerevisiae.oridb.org). In the present study, we divided ARSs on chromosome III into two groups [strongly active ARS (ARS305, ARS306, ARS307, ARS309, ARS310, and ARS315) and weakly active ARS (ARS300, ARS301/2/3, ARS320, ARS304, ARS308, ARS313, ARS314, ARS316, and ARS317/18)] according to ARS activity. To investigate the influence of ARS activity on gene expression, 25 loci adjacent to ARSs exhibiting different activities were selected on chromosome III, and RFP was integrated into these loci as a reporter by replacing the KanMX gene in strains from the YKO library (Fig. 1A). The relative fluorescence intensities of RFP were subsequently examined, with the results indicating that RFP exhibited distinct relative fluorescence intensities according to locus, ranging from 1.79 to 10.27 (Fig. 2A). Specifically, RFP inserted in close proximity to strongly active ARSs resulted in higher relative fluorescence intensity than that adjacent to weakly active ARSs. As shown in Fig. 2, the relative fluorescence intensity associated with RFP inserted near
Six strongly active ARSs in chromosome III were knocked out using CRISPR/Cas9 and substituted with the RFP cassette (Fig. 1C). We found that the relative fluorescence intensities associated with inserted RFP in most knockout strains (dARS305, dASR306, dARS307, dARS310, and dARS315) decreased relative to control strains (ARS305, ASR306, ARS307, ARS310, and ARS315). Among the knockout strains, ARS306 deletion (located in a compact region of chromosome III) resulted in the largest reduction in RFP expression (from 8.16 to 2.84) (Fig. 4A), whereas knockout of ARS309 (located in a relative loose region of chromosome III) did not notably affect RFP-related fluorescence (from 8.45 to 8.58). 3.4. The combined effect of chromosome structure and ARSs on gene expression The results suggested that gene expression was affected by
Fig. 2. The relative fluorescence intensity associated with RFP inserted nearby ARSs with different activities on chromosome III. (A) Tag height above the map line represents the degree of relative fluorescence intensity and the percentages beneath the map line indicated the ARS replication activities. Red and blue colors indicate strongly and weakly active ARSs on chromosome III, respectively. The centromere of chromosome III is denoted by a solid dot (orange). (B) The average relative fluorescence intensity associated with RFP inserted adjacent to loci near strongly active (red) and weakly active (blue) ARSs. *p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 3
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Fig. 3. The effect of ectopic expression of a strongly active ARS on gene expression in the yeast chromosome. The orange and grey bars represent the relative fluorescence intensities of RFP in the recombinant strains with or without strongly active ARS305, respectively. The X-axis shows the number of (A) weakly active or (B) strongly active ARSs along with RFP insertion in different recombinant strains. *p < 0.05, orange vs. grey bars.
ARSs did not enhance the expression of adjacent RFP genes, indicating no additive effect on ARS-related elevations in transcription. Moreover, ARS activity associated with DNA replication was also not additive. A previous study suggested that ARS activity inhibits other nearby ARSs, finding that when two ARSs were located in close proximity, only one ARS was activated in the yeast chromosome [33]. Another study reported that two strongly active ARSs separated by 6.5 k were capable of interfering with each other, with only one capable of initiating DNA replication, and the probability of both ARSs initiating replication at < 5% [4]. These findings suggest the likelihood that ARSs regulate gene expression through functions associated with DNA replication. The most important function of ARSs involve initiating DNA replication, where both loose and packed chromatin structures are transiently disrupted [34], thereby allowing transcription factors access to promoter regions [12]. Additionally, the chromatin-assembly process of newly replicated DNA facilitates transcription-factor access to important regulatory elements controlling gene transcription [35,36]. Therefore, ARSs might influence gene expression by promoting transcription-factor activity associated with the initiation of DNA replication. Some ARS elements share specific consensus sequences with the promoters of many genes. This includes the 5′-CGTnnnnnnnGA(G/C)-3′ sequence as the binding site of ARS-binding factor 1 [37], a multifunctional transcription factor involved in various nuclear events, including DNA replication [38,39], gene silencing [40], and transcription activation [35]. Although previous studies have not shown that ARSlike sequences in promoter regions can function as a strongly active ARSs, this concept represents a potential mechanism associated with the relationship between ARSs and gene expression. We knocked out six strongly active ARSs in order to examine the
chromosome structure. To eliminate the effect of chromosome structure on the gene expression, we inserted a cassette including RFP into pRS416 plasmids and replaced their original ARSs with four strongly active or four weakly active ARSs from the S. cerevisiae chromosome and used these to transform strain BY4742 (Fig. 1D). The strains with strongly active ARSs displayed higher relative fluorescence intensities than strains with weakly active ARSs, regardless of whether the ARS was located on the chromosome or in the plasmid (Fig. 5A and Supplementary Fig. 1). These results demonstrated that ARS activity was closely correlated with gene expression, regardless of DNA structure. Additionally, compared with other ARSs, ARS304 and ARS306 inserted into compact regions of chromosome III induced more changes in adjacent RFP expression between the chromosome and plasmid, indicating that a compact chromosome structure might restrict ARS function associated with gene expression. 4. Discussion Recent evidence suggests that ARSs affect gene expression; however, few studies have adequately explained the mechanism underlying this correlation. In this study, we found that reporter-gene expression was positively correlated with replication-related activities of adjacent ARSs in the yeast genome, demonstrating that ARS activity affected gene expression. A previous study reported that sequence direction was not a factor influencing gene expression in S. cerevisiae [17]. In the present study, we found a similar expression between two RFP genes inserted into the sense strand at the YCR036W site and the antisense strand at the YCR037C site adjacent to ARS313 (Fig. 2); the same result was observed for ARS317/18. Our results revealed that insertion of additional strongly active
Fig. 4. The effect of knocking out strongly active ARSs on gene expression in the yeast chromosome. (A) The grey and orange bars represent the relative fluorescence intensities of RFP in the recombinant strains with or without six strongly active ARSs. The X-axis shows the number of strongly active ARSs along with RFP insertion. *p < 0.05, orange vs. grey bars; **p < 0.001, orange vs. grey bars. (B) The negative Y-axis represents the contact frequency of the chromosome region where the ARSs are located, and the positive Yaxis represents the corresponding change in RFP-expression ratio between strains with or without ARSs in the chromosome. The ratio was calculated as follows: ratio = (ARS knockout − control) / control × 100%. The contact frequency of one ARS was calculated by summing the total contact frequencies of DNA fragments harboring the ARS with other fragments on chromosome III [41].
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Fig. 5. The influence of DNA structure on ARS-mediated gene expression. (A) Comparison of the effect of ARSs on gene expression between plasmid (orange bar) and chromosome (grey bar). *p < 0.05, orange vs. grey bars; **p < 0.001, orange vs. grey bars. (B) The negative Y-axis represents the contact frequency of the chromosome region where the ARS is located, and the positive Y-axis represents the corresponding change in RFP-expression ratio between strains harboring ARSs in the chromosome and in the plasmid. The ratio was calculated as follows: ratio = (ARS on plasmid − ARS on chromosome) / control × 100%. The contact frequency of one ARS was calculated by summing the total contact frequencies of DNA fragments harboring the ARS with other fragments on chromosome III [41].
roles of ARSs in gene expression, finding that fluorescence associated with RFP inserted adjacent to the strongly active ARSs mostly decreased, especially in the case of ARS306 deletion. By contrast, deletion of ARS309 had no effect on RFP fluorescence (Fig. 4A). The chromosome-conformation capture (3C) technique allows analysis of the spatial organization of chromatin in cells. Contact frequency is an important parameter of 3C technology and represents the interaction frequency between one segment and other segments while also indirectly reflecting the density of chromosome structure. Based on previous data concerning three-dimensional (3D) models of the yeast genome, we calculated local interactions of ARSs by analyzing the contact frequency of DNA regions harboring them relative to other fragments in the yeast chromosome [41]. The local interactions of chromosome regions near ARS306 was more extensive than those proximal to ARS309, suggesting that ARS306 was present in a more compact chromosome region than ARS309 (Fig. 4B). These results indicated that ARSs in compact chromosome regions exhibited stronger effects on gene expression than those in relaxed chromatin regions, thereby supporting the hypothesis that ARS elements might affect gene expression by providing a more open chromatin structure [15]. Chromosome structure influences gene expression, with a study of position-specific effects in humans reporting higher expression levels of genes located in regions displaying a more open chromatin structure [42]. To determine the possible effect of chromosome 3D structure on ARS function in gene expression, we compared changes in RFP-expression ratio between different locations of adjacent ARSs (in the chromosome and in the plasmid), with results showing small changes when the ARS was located in an unwound chromosome region (ARS308 and ARS309) and large changes when the ARS was located in compact chromosome regions (e.g., ARS304 and ARS306) (Fig. 5B). This supported our previous findings that ARSs affected gene expression according to chromosome structure. S. cerevisiae is a widely used host for expressing heterogeneous genes for synthetic biology applications and metabolic engineering; therefore, increasing attention has focused on the mechanisms that can regulate gene expression in S. cerevisiae. To this end, the findings of the present study confirmed a novel mechanism of gene regulation outside the gene promoter and highlighted the important effect of ARS activity on gene expression in S. cerevisiae.
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Funding Financial support was provided by the Ministry of Science and Technology of China (2014CB745100) and the National Natural Science Foundation of China (21390203, 21576198, 21622605, 21621004, and 31571358). 5
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
The effect of autonomously replicating sequences on gene expression in saccharomyces cerevisiae
T
Xiao-Le Wua,b, Yan-Hui Bia,b, Feng Gaoa,c, Ze-Xiong Xiea,b, Xia Lia,b, Xiao Zhoua,b, De-Jun Mad, ⁎ Bing-Zhi Lia,b, , Ying-Jin Yuana,b a
Frontier Science Center of Synthetic Biology (MOE), Key Laboratory of Systems Bioengineering (MOE), Tianjin University, Tianjin, 300072, China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China c Department of Physics, Tianjin University, Tianjin, 300072, China d State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China b
H I GH L IG H T S
replicating sequence (ARS) activity affected adjacent gene expression. • Autonomously active ARSs promoted gene expression in a non-synergistic manner. • Strongly of strongly active ARSs decreased adjacent gene expression. • Knockout • Chromosome structure influenced the effects of ARSs on gene expression.
A R T I C LE I N FO
A B S T R A C T
Keywords: Gene expression ARS Chromosome structure Synthetic biology Yeast
Autonomously replicating sequences (ARSs) are essential for genome replication and can regulate gene expression as potential cis-elements; however, the mechanisms underlying this regulation are unclear. In order to understand these mechanisms, we first replaced genes adjacent to ARSs on chromosome III of Saccharomyces cerevisiae with an expression cassette containing the gene encoding red fluorescence protein (RFP) and verified that the relative fluorescence intensity of RFP was positively correlated with the activities of the adjacent ARSs. Then, to investigate the effect of ARSs on gene expression, we designed ARS-insertion and -deletion variants, finding that the insertion of a strongly active ARS adjacent to a weakly active ARS enhanced adjacent gene expression, whereas the deletion of a strongly active ARS reduced the expression of the flanking RFP; however, insertion of multiple strong ARSs did not show a synergistic effect on gene expression. Further analysis suggested that ARSs located in compact chromosomal regions exerted a stronger effect on gene expression than those in relaxed chromosomal regions. These results demonstrated that ARSs regulated S. cerevisiae gene expression by interacting with the chromosome structure and provide a potential strategy for regulating gene expression in synthetic biology and metabolic engineering studies using S. cerevisiae.
1. Introduction Unlike prokaryotes, the initiation of DNA replication usually occurs at discrete chromosomal loci in yeast and higher eukaryotes [1–3]. In the eukaryotic cell cycle, replication is activated at different time points [4]; previous studies identified autonomously replicating sequence
(ARS) elements in budding yeast Saccharomyces cerevisiae as key elements involved in initiating DNA replication and maintaining genome stability [5–7]. Further studies indicated that ARS initiation time and efficiency varied in the yeast chromosome [8–12]. A previous study examined the ARS activity by analyzing the replication origin efficiency of DNA fragments carrying the ARS elements [13]. The different shapes
Abbreviations: 2D, two dimensional; 3D, three dimensional; ARS, autonomously replicating sequence; gRNA, genomic RNA; MAR, matrix attachment region; PCR, polymerase chain reaction; rARS, ribosomal ARS; RFP, red fluorescent protein; YKO, yeast knockout ⁎ Corresponding author at: Frontier Science Center of Synthetic Biology (MOE), Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China. E-mail address: [email protected] (B.-Z. Li). https://doi.org/10.1016/j.bej.2019.107250 Received 21 February 2019; Received in revised form 27 May 2019; Accepted 28 May 2019 Available online 29 May 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
Biochemical Engineering Journal 149 (2019) 107250
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created by adding 20 mg/L uracil to SC-Leu-Ura media and SC-Ura media was created by adding 20 mg/L leucine to SC-Leu-Ura media [25]. Amino acids were purchased from Genview (Beijing, China).
of replication intermediates could be separated by two-dimensional (2D) electrophoresis. The "bubble-shaped" replication intermediates represented an active origin and "Y-shaped" intermediates reflected a passive replication by a fork from an origin external to the fragment [14]. The replication origin activity was revealed by the occurrence of bubble shape replication intermediates, which varied from 90% to < 10% [9,10]. Recently, increasing evidence has shown that ARSs are associated with adjacent gene expression [15]. The integration of heterogeneous genes at different loci in the yeast genome resulted in significant differences in gene-expression levels [16–18], suggesting that ARSs might be a factor affecting gene expression. Upon subsequent random insertion of the heterogeneous genes at 20 loci in the yeast genome, the highest transcriptional activity was observed when the reported gene was inserted within 400 bp of the closest ARS [19]. Matrix attachment regions (MARs) in higher eukaryotes, such as Drosophila [20] and fish [21], exhibit similar functions involving DNA replication as ARSs in the yeast, with one study demonstrating that the MARs integrated within an S. cerevisiae plasmid affected the expression of enhanced green fluorescent protein [21]. Additionally, the replication activity of an ARS might be a prerequisite for regulating gene expression. A study of a ribosomal ARS (rARS) demonstrated that ribosomal RNA genes flanking activated rARSs were preferentially active in S. cerevisiae [15], and our previous study indicated that only reporter genes close to strongly active ARSs exhibited high expression levels [22]. Our previous study investigated ARS functions on gene expression in situ [22]. Therefore, in the present study, we ectopically integrated strongly active ARSs on chromosome III of S. cerevisiae in order to examine their effects on the expression of nearby genes. Additionally, we knocked out five strongly active ARSs and explored the effects on the expression of adjacent genes, with subsequent analyses of ARS-specific effects on gene expression in both the chromosome and a plasmid.
2.2. Construction of expression cassettes We used RFP as the reporter gene with the pURA3 promoter cloned from an existing plasmid maintained in our lab. The homology arms (kanMX-L and kanMX-R) were cloned from the genome of the YKO collection, and ARS305 was cloned from the genome of BY4742. DNA fragments were integrated into the pUC19 plasmid and cloned for the subsequent transformation procedures. 2.3. Construction of guide RNA expression plasmids A genomic RNA (gRNA)-expression plasmid targeting a specific genomic locus was constructed according to a previous method [26,27]. First, the pRS426-SNR52p-gRNA-SUP4t plasmid (stored in our lab) was digested by NotI (New England Biolabs, Ipswich, MA, USA) and purified by agarose gel electrophoresis. Second, the oligonucleotide primers were annealed for gRNA-expression plasmid construction. The primers included a 20-bp gRNA fragment and 40-bp overlapping sequence, with pRS426-SNR52p-gRNA-SUP4t located at the cleavage site. Third, the 60-bp fragment and linear plasmid were connected through Gibson assembly (New England Biolabs, Ipswich, MA, USA) and transformed into Escherichia coli [28]. 2.4. CRISPR/Cas9-based transformation A CRISPR/Cas9-based transformation strategy was used to substitute the kanMX cassettes and ARS, as described previously [29]. First, the Cas9-expression plasmid constructed from plasmid pRS415 was transformed into strains, followed by incubation on SC-Leu plates at 30 °C for 2 days [26]. Polymerase chain reaction (PCR) was used to confirm the correct strains. Second, we transformed both the gRNA plasmid and the DNA fragment into the strain, with SC-Leu-Ura medium used as a screening medium. After completion of the transformation, cells were plated and incubated for 2 days at 30 °C, and PCR was used to verify candidate colonies.
2. Materials and methods 2.1. Strains and media Recombinant strains were derived from a yeast knockout (YKO) collection using homologous recombination [23,24] and are listed in Supplementary Table 1. The media included SC-Leu-Ura, SC-Leu, and SC-Ura. SC-Leu-Ura was a synthetic dextrose medium containing 6.7 g/L yeast nitrogen base without amino acids (Solarbio, Beijing, China), 20 g/L glucose, 2 g/L complete supplement mixture (without histidine, leucine, tryptophan, uracil), 20 mg/L histidine, and 20 mg/L tryptophan. SC-Leu media was
2.5. RFP expression in plasmids Plasmids carrying different ARSs were constructed based on the pRS416 plasmid [30]. First, we used PCR to obtain the pRS416 fragment lacking the ARS, followed by cloning of the ARSs in chromosome Fig. 1. Homologous recombination and vector configuration. (A) The RFP gene was integrated into loci adjacent to ARSs on chromosome III by replacing the kanMX gene. The DNA fragment used included RFP with the pURA3 promoter and two homology arms (KanMX-L and KanMX-R). (B) Strongly active ARS305 together with RFP were integrated into loci adjacent to ARSs on chromosome III. The DNA fragment included ARS305, RFP with pURA3, and two homology arms (kanMX-L and kanMX-R). (C) Knockout of strongly active ARS (ARSa) on chromosome III and insertion of RFP at loci adjacent to the deleted ARSs. Two DNA fragments were used: one included the homologous arms on both sides of the ARS, and the other was the same as the fragment used in (A). (D) The ARS fragment in the pRS416 plasmid was replaced with four strongly active or four weakly active ARSs, followed by insertion of RFP. 2
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strongly active ARSs was > 7 (average intensity: 8.56), which was significantly higher than the average relative fluorescence intensity (4.24) associated with RFP inserted adjacent to weakly active ARSs (Fig. 2B).
III from the yeast genome. We then performed Gibson assembly to connect the ARS fragment to the plasmid lacking the ARS (Fig. 1D), followed by transformation of the BY4742 strain with the recombinant plasmids, respectively, with SC-Ura medium used as a screening medium. After transformation, cells were plated and incubated for 2 days at 30 °C, and PCR was preformed used to confirm successful transformation.
3.2. Strongly active ARSs affected gene expression in a dominant rather than additive manner To further investigate the effect of ARS activity on gene expression, the strongly active ARS305 adjacent to RFP was integrated into loci adjacent to other ARSs on chromosome III, including six strongly active and eight weakly active ARSs (Fig. 1B). We found that the expression of RFP inserted near weakly active ARSs, except ARS304 and ARS313, was significantly elevated due to the ARS305 insertion, with the relative fluorescence intensity associated with RFP inserted adjacent to ARS300, ARS301/302/303, ARS320, ARS308, ARS316, and ARS317/318 increasing from 1.79, 3.51, 2.99, 4.71, 5.82, and 3.03 to 4.49, 5.42, 4.87, 6.98, 8.04, and 9.75, respectively (Fig. 3A). However, insertion of ARS305 did not enhance the fluorescence of RFP inserted adjacent to strongly active ARSs, except ARS 315 (from 9.81 to 11.37), suggesting that ARS-related influence on gene expression was not synergetic (Fig. 3B).
2.6. Fluorescence assays Selected strains harboring the reporter gene were grown overnight and resuspended to an absorbance at 600 nm of 0.2, followed by incubating at 30 °C for 4 h. Yeast cells were harvested by centrifugation at 4000 × g for 2 min and resuspended in phosphate-buffered saline. Fluorescence assays were performed individually in cuvettes, and RFP fluorescence was measured using a SpectraMax M2 detector (Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 587 nm and an emission wavelength of 610 nm [31,32]. Relative fluorescence intensity was derived as fluorescence/optical density at 600 nm. 3. Results 3.1. Positive relationship between gene expression and ARS activity
3.3. Knockout of strongly active ARSs decreased gene expression
There are 20 ARSs on chromosome III of S. cerevisiae according to the DNA Replication Origin Database (http://cerevisiae.oridb.org). In the present study, we divided ARSs on chromosome III into two groups [strongly active ARS (ARS305, ARS306, ARS307, ARS309, ARS310, and ARS315) and weakly active ARS (ARS300, ARS301/2/3, ARS320, ARS304, ARS308, ARS313, ARS314, ARS316, and ARS317/18)] according to ARS activity. To investigate the influence of ARS activity on gene expression, 25 loci adjacent to ARSs exhibiting different activities were selected on chromosome III, and RFP was integrated into these loci as a reporter by replacing the KanMX gene in strains from the YKO library (Fig. 1A). The relative fluorescence intensities of RFP were subsequently examined, with the results indicating that RFP exhibited distinct relative fluorescence intensities according to locus, ranging from 1.79 to 10.27 (Fig. 2A). Specifically, RFP inserted in close proximity to strongly active ARSs resulted in higher relative fluorescence intensity than that adjacent to weakly active ARSs. As shown in Fig. 2, the relative fluorescence intensity associated with RFP inserted near
Six strongly active ARSs in chromosome III were knocked out using CRISPR/Cas9 and substituted with the RFP cassette (Fig. 1C). We found that the relative fluorescence intensities associated with inserted RFP in most knockout strains (dARS305, dASR306, dARS307, dARS310, and dARS315) decreased relative to control strains (ARS305, ASR306, ARS307, ARS310, and ARS315). Among the knockout strains, ARS306 deletion (located in a compact region of chromosome III) resulted in the largest reduction in RFP expression (from 8.16 to 2.84) (Fig. 4A), whereas knockout of ARS309 (located in a relative loose region of chromosome III) did not notably affect RFP-related fluorescence (from 8.45 to 8.58). 3.4. The combined effect of chromosome structure and ARSs on gene expression The results suggested that gene expression was affected by
Fig. 2. The relative fluorescence intensity associated with RFP inserted nearby ARSs with different activities on chromosome III. (A) Tag height above the map line represents the degree of relative fluorescence intensity and the percentages beneath the map line indicated the ARS replication activities. Red and blue colors indicate strongly and weakly active ARSs on chromosome III, respectively. The centromere of chromosome III is denoted by a solid dot (orange). (B) The average relative fluorescence intensity associated with RFP inserted adjacent to loci near strongly active (red) and weakly active (blue) ARSs. *p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 3
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Fig. 3. The effect of ectopic expression of a strongly active ARS on gene expression in the yeast chromosome. The orange and grey bars represent the relative fluorescence intensities of RFP in the recombinant strains with or without strongly active ARS305, respectively. The X-axis shows the number of (A) weakly active or (B) strongly active ARSs along with RFP insertion in different recombinant strains. *p < 0.05, orange vs. grey bars.
ARSs did not enhance the expression of adjacent RFP genes, indicating no additive effect on ARS-related elevations in transcription. Moreover, ARS activity associated with DNA replication was also not additive. A previous study suggested that ARS activity inhibits other nearby ARSs, finding that when two ARSs were located in close proximity, only one ARS was activated in the yeast chromosome [33]. Another study reported that two strongly active ARSs separated by 6.5 k were capable of interfering with each other, with only one capable of initiating DNA replication, and the probability of both ARSs initiating replication at < 5% [4]. These findings suggest the likelihood that ARSs regulate gene expression through functions associated with DNA replication. The most important function of ARSs involve initiating DNA replication, where both loose and packed chromatin structures are transiently disrupted [34], thereby allowing transcription factors access to promoter regions [12]. Additionally, the chromatin-assembly process of newly replicated DNA facilitates transcription-factor access to important regulatory elements controlling gene transcription [35,36]. Therefore, ARSs might influence gene expression by promoting transcription-factor activity associated with the initiation of DNA replication. Some ARS elements share specific consensus sequences with the promoters of many genes. This includes the 5′-CGTnnnnnnnGA(G/C)-3′ sequence as the binding site of ARS-binding factor 1 [37], a multifunctional transcription factor involved in various nuclear events, including DNA replication [38,39], gene silencing [40], and transcription activation [35]. Although previous studies have not shown that ARSlike sequences in promoter regions can function as a strongly active ARSs, this concept represents a potential mechanism associated with the relationship between ARSs and gene expression. We knocked out six strongly active ARSs in order to examine the
chromosome structure. To eliminate the effect of chromosome structure on the gene expression, we inserted a cassette including RFP into pRS416 plasmids and replaced their original ARSs with four strongly active or four weakly active ARSs from the S. cerevisiae chromosome and used these to transform strain BY4742 (Fig. 1D). The strains with strongly active ARSs displayed higher relative fluorescence intensities than strains with weakly active ARSs, regardless of whether the ARS was located on the chromosome or in the plasmid (Fig. 5A and Supplementary Fig. 1). These results demonstrated that ARS activity was closely correlated with gene expression, regardless of DNA structure. Additionally, compared with other ARSs, ARS304 and ARS306 inserted into compact regions of chromosome III induced more changes in adjacent RFP expression between the chromosome and plasmid, indicating that a compact chromosome structure might restrict ARS function associated with gene expression. 4. Discussion Recent evidence suggests that ARSs affect gene expression; however, few studies have adequately explained the mechanism underlying this correlation. In this study, we found that reporter-gene expression was positively correlated with replication-related activities of adjacent ARSs in the yeast genome, demonstrating that ARS activity affected gene expression. A previous study reported that sequence direction was not a factor influencing gene expression in S. cerevisiae [17]. In the present study, we found a similar expression between two RFP genes inserted into the sense strand at the YCR036W site and the antisense strand at the YCR037C site adjacent to ARS313 (Fig. 2); the same result was observed for ARS317/18. Our results revealed that insertion of additional strongly active
Fig. 4. The effect of knocking out strongly active ARSs on gene expression in the yeast chromosome. (A) The grey and orange bars represent the relative fluorescence intensities of RFP in the recombinant strains with or without six strongly active ARSs. The X-axis shows the number of strongly active ARSs along with RFP insertion. *p < 0.05, orange vs. grey bars; **p < 0.001, orange vs. grey bars. (B) The negative Y-axis represents the contact frequency of the chromosome region where the ARSs are located, and the positive Yaxis represents the corresponding change in RFP-expression ratio between strains with or without ARSs in the chromosome. The ratio was calculated as follows: ratio = (ARS knockout − control) / control × 100%. The contact frequency of one ARS was calculated by summing the total contact frequencies of DNA fragments harboring the ARS with other fragments on chromosome III [41].
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Fig. 5. The influence of DNA structure on ARS-mediated gene expression. (A) Comparison of the effect of ARSs on gene expression between plasmid (orange bar) and chromosome (grey bar). *p < 0.05, orange vs. grey bars; **p < 0.001, orange vs. grey bars. (B) The negative Y-axis represents the contact frequency of the chromosome region where the ARS is located, and the positive Y-axis represents the corresponding change in RFP-expression ratio between strains harboring ARSs in the chromosome and in the plasmid. The ratio was calculated as follows: ratio = (ARS on plasmid − ARS on chromosome) / control × 100%. The contact frequency of one ARS was calculated by summing the total contact frequencies of DNA fragments harboring the ARS with other fragments on chromosome III [41].
roles of ARSs in gene expression, finding that fluorescence associated with RFP inserted adjacent to the strongly active ARSs mostly decreased, especially in the case of ARS306 deletion. By contrast, deletion of ARS309 had no effect on RFP fluorescence (Fig. 4A). The chromosome-conformation capture (3C) technique allows analysis of the spatial organization of chromatin in cells. Contact frequency is an important parameter of 3C technology and represents the interaction frequency between one segment and other segments while also indirectly reflecting the density of chromosome structure. Based on previous data concerning three-dimensional (3D) models of the yeast genome, we calculated local interactions of ARSs by analyzing the contact frequency of DNA regions harboring them relative to other fragments in the yeast chromosome [41]. The local interactions of chromosome regions near ARS306 was more extensive than those proximal to ARS309, suggesting that ARS306 was present in a more compact chromosome region than ARS309 (Fig. 4B). These results indicated that ARSs in compact chromosome regions exhibited stronger effects on gene expression than those in relaxed chromatin regions, thereby supporting the hypothesis that ARS elements might affect gene expression by providing a more open chromatin structure [15]. Chromosome structure influences gene expression, with a study of position-specific effects in humans reporting higher expression levels of genes located in regions displaying a more open chromatin structure [42]. To determine the possible effect of chromosome 3D structure on ARS function in gene expression, we compared changes in RFP-expression ratio between different locations of adjacent ARSs (in the chromosome and in the plasmid), with results showing small changes when the ARS was located in an unwound chromosome region (ARS308 and ARS309) and large changes when the ARS was located in compact chromosome regions (e.g., ARS304 and ARS306) (Fig. 5B). This supported our previous findings that ARSs affected gene expression according to chromosome structure. S. cerevisiae is a widely used host for expressing heterogeneous genes for synthetic biology applications and metabolic engineering; therefore, increasing attention has focused on the mechanisms that can regulate gene expression in S. cerevisiae. To this end, the findings of the present study confirmed a novel mechanism of gene regulation outside the gene promoter and highlighted the important effect of ARS activity on gene expression in S. cerevisiae.
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Funding Financial support was provided by the Ministry of Science and Technology of China (2014CB745100) and the National Natural Science Foundation of China (21390203, 21576198, 21622605, 21621004, and 31571358). 5
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