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Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii Elvira R. Eivazova, Sergei A. Markov* Biology Department, Austin Peay State University, P.O. Box 4718, Clarksville, TN 37041, USA
article info
abstract
Article history:
The direct relationship between hydrogenase gene conformation and its function in green
Received 23 July 2012
alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation
Received in revised form
in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of
6 September 2012
C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture
Accepted 13 September 2012
technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene
Available online 11 October 2012
region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the
Keywords:
gene region is present.
Biohydrogen
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
[FeFe]-hydrogenase gene
reserved.
Gene expression Chromosome conformation capture (3C) Chlamydomonas reinhardtii
1.
Introduction
Molecular hydrogen (H2) is one of the possible energy carriers of the future [1]. It is considered a fuel with low environmental impact since its combustion product (water) is non-polluting. Hydrogen is a renewable form of energy; it can be produced from water again. The conventional industrial methods for H2 production are costly and the problem has been to find a cheaper way to produce hydrogen. Biological H2 (biohydrogen) production by algal species has a number of advantages and it could be a cost effective alternative to the current industrial methods of H2 production [2]. Microscopic green algae produce H2 in photosynthetic reactions from water using sunlight as an energy source. Hydrogen production based on green algae holds the promise of generating
a renewable hydrogen fuel due to availability of large amounts of solar light and water. Green algae utilize the enzyme hydrogenase, which catalyzes H2 production, but only under anaerobic conditions, because the enzyme is very sensitive to O2 [3,4]. In addition, the hydrogenase gene is not expressed in the presence of O2 [5], but is rapidly expressed during anaerobic adaptation of the cells. Molecular mechanisms that regulate the hydrogenase gene expression under anaerobic conditions are poorly understood. Experimental evidence was provided recently that expression of a gene or gene family can be regulated through looping and bending of chromosomes [6e8]. Therefore, we expect that a hydrogenase gene region in green algae may have a spatial configuration that will have a considerable effect on gene activation, thus providing a mechanism for gene regulation.
* Corresponding author. Tel.: þ1 931 221 7440; fax: þ1 931 221 6323. E-mail addresses: [email protected] (E.R. Eivazova), [email protected] (S.A. Markov). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2012.09.087
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
Our hypothesis was that the hydrogenase gene in green algae adopts a specific conformation required for gene expression. Changes in such conformation provide the means for transcriptional activation of the hydrogenase genes under anaerobic conditions or their silencing under aerobic conditions. The gene conformation was assessed by the chromosome conformation capture (3C) assay, which is designed to estimate the interactions between genomic regions in vivo and to model a 3-D chromosome organization in the gene [9]. The basic principle of this method is that intact nuclei are isolated from cultured live cells and subjected to paraformaldehyde cross-linking to fix those segments of genomic DNA (via attached proteins) that are in close physical proximity to each other (Fig. 1). Cross-linked genomic DNA is digested with a restriction enzyme of choice and ligated at very low DNA concentrations that favor intramolecular interactions. Products of ligation are analyzed by PCR using primers designed for each restriction site to test for possible interactions between the restriction sites. The presence of a specific PCR product would indicate relative proximity of any two restriction sites to one another captured at a given point in time. We studied the microscopic eukaryotic organism, green alga Chlamydomonas reinhardtii. In order to implement 3C method, the whole genomic sequence of the experimental organism is required. A draft of the entire genome of Chlamydomonas reinhardtii CC-503 cw92 (mtþ) became available in 2004 and annotated in 2008. Green algae, along with some anaerobic bacteria and protozoa, possess [FeFe]-hydrogenases. The [FeFe]hydrogenase gene, hydA1 (referred to as HydA1 in some publications) from C. reinhardtii has been isolated and characterized [3]. A second [FeFe]-hydrogenase gene, hydA2, was identified in C. reinhardtii in 2003 [10]. It was reported that [FeFe]hydrogenase genes are transcriptionally regulated by presence or absence of O2 and by other unknown factors [11]. Therefore, a better understanding of the fundamental biology
17789
of hydrogenase gene (hydA1) expression would help the future development of economically viable systems for H2 production.
2.
Materials and methods
2.1.
Cell culture
The Chlamydomonas CC-503 cw92 (mtþ) cell wall-less strain was obtained from Dr. Elizabeth Harris (Chlamydomonas Genetics Center, Duke University). It was grown in a Triseacetateephosphate medium, pH 7.0, in 250-mL Erlenmeyer flasks with a cell suspension volume of 100 mL [12]. Continuous light was provided by cool white fluorescent lamps (15 mmol m2 s1 on the surface of the culture). Threeday-old cultures of green algae were used in experimental procedures. Cells were harvested at OD665 w 0.45 corresponding to a chlorophyll concentration of w17 mg ml1.
2.2.
Hydrogen production
Samples of cell suspension (3 mL) were vacuum degassed (270e300 torr) and flashed with N2 to induce anaerobiosis. Following this procedure, the samples were incubated under illumination provided by cool white fluorescent lamps (15 mmol m2 s1 on the surface of the culture) for 12 h in sealed 15 mL vials fitted with rubber stoppers. Hydrogen production was measured using a Gow-Mac gas chromatograph (Bethlehem, PA) equipped with a molecular sieve 5A column and a thermal conductivity detector. Nitrogen was used as a carrier gas.
2.3.
3C assay
We used the 3C assay [9] with some modifications.
2.3.1.
Nuclei isolation
The assay was performed with nuclei isolated from three-dayold algal cultures. To create anaerobic conditions, some of the cell cultures were additionally anaerobically incubated in sealed Erlenmeyer flasks under N2 atmosphere for 12 h in order to induce the expression of the hydrogenase gene. Nuclei were isolated by the modified method of Keller et al. [13]. Before nuclear isolation, cells were diflagellated by pH shock. A protease inhibitor was added (Pefabloc from Sigma, St. Louis, MO, 10 mL/mL), followed by 1% Triton X-100 (Sigma, St. Louis, MO) to lyse the cells. Nuclei were identified and counted in the lysed cell material using the fluorescent DNAbinding dye SYBR green from Molecular Probes, Eugene, OR (1:10,000 dilution) and a Nikon fluorescence microscope (Japan) equipped with the Nikon Digital Camera DMX1200F.
2.3.2. Fig. 1 e Schematic representation of the 3C methodology: formaldehyde-induced DNA attachment via protein, AatII restriction enzyme digestion, and PCR-mediated detection of ligation products after reversal of the cross-links (protein removal).
Formaldehyde cross-linking of proteins and DNA
Formaldehyde (Sigma St. Louis, MO) was added directly to isolated intact nuclei to a final concentration of 1% in a buffer to cross-link proteins and DNA. Nuclei were incubated and gently mixed for optimal cross-linking; after 10 min glycine (0.125 M final concentration) was added to stop the reaction. To remove non-cross-linked proteins from DNA, SDS (Sigma
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
St. Louis, MO) was added to a final concentration of 0.1% for 10 min, followed by Triton X-100 at concentration of 1%.
2.3.3.
Restriction digestion
Cross-linked DNA was digested with the AatII restriction enzyme (New England Biolabs, Beverly, MA). The restriction digestion was optimized by varying the concentration of enzymes and the reaction volumes. Digestion was most successful at a final volume of 50 mL with 3 mL of enzyme and 10 mL of DNA. The incubation time for digestion of chromatin was 1 h at 37 C. The mixture was incubated with 1.6% SDS for 20 min at 65 C to inactivate AatII.
2.3.4.
Ligation
To favor intermolecular interactions the DNA material was ligated at a low DNA concentration. We established optimal conditions for ligation (T4 ligase, New England Biolabs) by using DNA at different concentrations and monitoring products of ligation by PCR. DNA was ligated for 1 h at 16 C. After ligation, the mixture was incubated with 1.6% SDS for 20 min at 65 C to inactivate AatII.
2.3.5.
Reverse cross-linking
Cross-linking of proteins and DNA was reversed by overnight incubation of the samples with proteinase K (Promega, Madison, WI) at 65 C.
2.3.6.
DNA purification
five restrictions sites. Primer pairs homologous (1þ1, 2þ2, 3þ3, 4þ4, 5þ5) that do not showed any PCR products were eliminated from consideration (from our further experiments). For example, we had primers B5 and B6 and B7 and B8 for our restriction site 1 (Fig. 2). Primers B7 and B8 did not show any PCR products and were never used again. Forward and reverse primers for PCR amplification were used in all pairwise homologous (1þ1, 2þ2, 3þ3, 4þ4, 5þ5) and nonhomologous (1þ2, 2þ1, 1þ3, 3þ1, 1þ4, 4þ1, 1þ5, 5þ1, etc.) combinations. In other words, every forward primer for one restriction site was combined in pairs with a reverse primer of every other restriction site in order to complete the set of all possible primer pair combinations. The PCR products were resolved by electrophoresis on 2% agarose gels.
3.
Results
3.1. Hydrogen production as a measure of hydA1 gene expression To find out when hydrogenase gene is expressed, we measured H2 production by C. reinhardtii cells during several days. Three- and four-day-old cultures showed significant levels of H2 production under anaerobic conditions (Fig. 3). We did not detect any H2 production under aerobic conditions. The three-day-old cultures with the maximal H2 production were used in the gene conformation experiments (3C assay).
DNA was extracted by standard “phenolechloroform” method.
3.2. 2.3.7. Analysis of interactions between restriction sites in hydrogenase gene region Primer pairs were designed (and synthesized by Sigma Genosys, Woodlands, TX) for each of the five AatII restriction sites (Fig. 2) positioned along the 29-kb hydrogenase gene region to make approximately 250 bp long DNA products. Originally, two pairs of primers were designed for each of the 159990 bp
130396 bp Base position Chlamydomonas hydA1
Search for the gene (hydA) on C. reinhardtii DNA
To perform the 3C assay (DNA conformation assay) the hydA1 gene location on C. reinhardtii DNA must be known. Using the database from the Joint Genome Institute (http://www.jgi.doe. gov) and sequences of a hydrogenase gene and its promoter from Genbank (accession numbers AJ012098 and AJ308413), the whole genome sequence of C. reinhardtii has been searched. The [FeFe]-hydrogenase gene (hydA1) region (29 kb) has been located in contigs 20 and 21 (scaffold 12) within a base position range of 130396 and 159990 (Fig. 2). The position of the promoter was located manually using the Chlamydomonas whole DNA sequence and hydA1 data deposited in Genbank (accession numbers above) by Happe and Kaminski [3].
AatII sites 1
2
3
4
5
Fig. 2 e Schematic representation of the 29-bp hydA1 (gene) region on Chlamydomonas chromosome. Locations of AatII restriction sites are shown. Base count is indicated according to the US Department of Energy Joint Genome Institute website (http://www.jgi.doe.gov). Positions of the five restriction sites from the start of the hydA1 promoter as follows: site 1, L10206; site 2, L7049; site 3, L2136; site 4, D498; site 5, D15055. The bolded area in a Chlamydomonas hydA1 gene indicates the promoter region.
Fig. 3 e Hydrogen production by Chlamydomonas reinhardtii as a function of time. A e H2 production under anaerobic conditions; - e H2 production under aerobic conditions.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
3.3.
Search for the restriction enzyme
The gene conformation 3C methodology required to perform DNA restriction enzyme digestion (Fig. 1). The specific restriction enzyme is needed which can cut C. reinhardtii DNA several times in hydA1 gene region and, at least one time, in directly within gene promoter [7,9]. DNA fragments after restriction digestion should be around 3000 bp in size. Using the Webcutter2 (http://rna.lundberg.gu.se/cutter2) we found a restriction enzyme, AatII, which produced five restriction sites in the hydA1 gene region of C. reinhardtii DNA (Fig. 2) along the 29 kb gene region. Several other enzymes were considered for our experiments such as NotI. Compared to AatI, NotI can make only 3 cuts in the 29 kilobase (kb) chromosome region of our interest.
3.4.
Application of 3C methodology
3.4.1.
Nuclei isolation
The first step in 3C assay was to isolate nuclei. The method employed for nuclei isolation allowed us to visualize in real time the release of nuclei from algal cells treated with detergent (Fig. 4). Isolated nuclei were uniform in size and their appearance was similar to nuclei of intact cells.
3.4.2. Formaldehyde cross-linking of proteins and DNA, restriction digestion and DNA ligation Isolated nuclei were subjected to formaldehyde treatment to fix DNA sites that are in close physical proximity to each other. Cross-linked DNA was digested with AatII restriction enzyme and DNA fragments were ligated at very low DNA concentrations that favor intermolecular interactions.
3.5. Specific interaction between restriction enzyme sites: gene conformation under aerobic and anaerobic conditions Availability of cross-linked DNA ligation products were assessed using PCR using primers to AatII restriction sites in all possible combinations. As a control, we digested non-crosslinked DNA (not subject to formaldehyde treatment) with restriction enzyme AatII followed by ligation and PCR. We
Fig. 4 e Nuclei pop out of Chlamydomonas cells after their treatment with 1% Triton X-100. Picture taken using Nikon fluorescence microscope equipped with the Nikon Digital Camera.
17791
followed the general control guidelines described by Dekker [14]. Representative PCR products with homologous primers (5þ5) and non-homologous primers (2þ5, 3þ5) from crosslinked DNA of aerobically grown algae are shown in Fig. 5A. The obtained experimental results allow us to conclude that restriction site 5 interacts with sites 2 and 3. Because of the presence of PCR products obtained with the primers specific for restriction sites, located several kilobase (kb) away from each other, we reason that these sites are positioned in close proximity by looping of chromosome. Thus, these data provided important evidence for a loop configuration in the hydrogenase gene region under aerobic conditions (Fig. 6A). In DNA templates obtained under anaerobic conditions we did not observe any interactions between the restriction sites, thus there was no any loop organization in the hydrogenase gene region (Figs. 5 and 6B).
4.
Discussion
Gene expression is regulated at different levels in the pathway from gene to functional protein. For most genes the initiation of transcription is the most important point of control [15]. Our understanding of gene regulation is moving from a one-dimensional picture of regulatory DNA sequences (“sending” proteins to express or silence genes) into a threedimensional spatial organization of chromosomes influencing gene expression [16e18]. Physical looping of DNA with the help of proteins permits gene expression or silencing in eukaryotes and, possibly, in prokaryotic microorganisms [19]. With the introduction of chromosome conformation capture (3C) methodology [9] it has now become possible to study the role of chromosome conformation in the course of gene expression or silencing. This method gives a new dimension to the study of gene regulation by determining the cross-linking frequencies of interaction between different chromosome segments within a gene region in vivo, and to model chromosome conformation before and after gene expression. This approach was successfully applied in a mammalian system to interferon gamma gene, Ifng [7]. The chromatin conformation of the Ifng gene was analyzed in T cell. The results showed a looping structure, which changed upon the Ifng expression during T cell differentiation. In such multi-looped conformation, several distal regions were brought into close proximity to the promoter region, forming a so-called “chromatin hub” configuration. We have analyzed chromosome conformation in the 29 kilobase (kb) region containing [FeFe]-hydrogenase gene
Fig. 5 e A & B. Representative PCR products generated with cross-linked DNA templates of C. reinhardtii cultured aerobically (A) and anaerobically (B).
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
regulation of gene expression. The results showed a loop organization within the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. We reasoned that because DNA is folded within the hydrogenase gene region the enzyme RNA-polymerase cannot transcribe the [FeFe]-hydrogenase gene. In contrast, under anaerobic conditions, when the [FeFe]-hydrogenase gene is active (expressed), no loop conformation in the gene region was found. This study also identified functionally significant fragments in the hydrogenase gene (hydA1) region. In future work, these fragments could be selectively deleted in order to express the hydrogenase gene, even in the presence of molecular oxygen.
Acknowledgments This work was supported by National Science Foundation (USA), award # 0821571 and Austin Peay State University and DePauw Universities Summer Faculty Research Programs. We thank Ms. Brittany Durr for valuable assistance with the experimental work.
Fig. 6 e [FeFe]-hydrogenase gene region of a green alga Chlamydomonas reinhardtii under aerobic (A) and anaerobic conditions (B).
(hydA1) of C. reinhardtii by 3C technique under aerobic and anaerobic conditions. The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions (Fig. 6A). In this loop conformation, parts of the chromosome around the hydrogenase gene were brought into close spatial proximity to each other, thus effectively silencing this gene. Chromosome conformation of the hydrogenase gene region changed to “unfold configuration” (Fig. 6B) upon transition of cells to anaerobic conditions which provided the means for transcriptional activation of the hydrogenase gene and subsequent H2 production. The observed correlation between hydA1 gene activity and its conformation implies the regulatory mechanism for hydrogenize gene activation and silencing by the sequential changes in its spatial configuration. Our data are important to advancing knowledge and understanding of hydrogenase gene expression, because it presented for the first time the experimental evidence of the conformational regulation of such gene expression in algae. Better understanding of the regulation of hydrogenase genes will help to optimize H2 production by microscopic algae and other microorganisms. New strains of algae that have highly expressed hydrogenase genes could be engineered. This research is also contributed to our general understanding of mechanisms of gene regulation in living organisms.
5.
Conclusions
The data presented in this paper for the first time showed the mechanism of [FeFe]-hydrogenase gene (hydA1) expression in the green alga C. reinhardtii, or as we called it, the conformational
references
[1] Cammack R, Frey M, Robson RL. Hydrogen as fuel: learning from nature. London: Taylor & Francis; 2001. lu TN. Hydrogen production by biological [2] Das D, Vezirog processes: a survey of literature. Int J Hydrogen Energy 2001; 26:13e28. [3] Happe T, Kaminski A. Differential regulation of the Fehydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. Eur J Biochem 2002;269:1022e32. [4] Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wu¨nschiers R, Lindblad P. Hydrogenases and hydrogen metabolism of cyanobacteria. MMBR 2002;66:1e20. [5] Girbal L, von Abendroth G, Winkler M, Benton PMC, MeynialSalles I, Croux C, et al. Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 2005;71:2777e81. [6] Tolhuis B, Palstra R-J, Splinter E, Grosveld F, Laat W. Looping and interaction between hypersensitive sites in the active b-globin locus. Mol Cell 2002;10:1453e65. [7] Eivazova ER, Aune TM. Dynamic alterations in the conformation of the Ifng gene region during T helper cell differentiation. PNAS 2004;101:251e6. [8] Razin SV, Iarovaia OV, Sjakste N, Sjakste T, Bagdoniene L, Rynditch AV, et al. Chromatin domains and regulation of transcription. J Mol Biol 2007;369:597e607. [9] Dekker J, Rippe K, Dekker M, Kleckner N. Capturing chromosome conformation. Science 2002;295:1306e11. [10] Forestier M, King P, Zhang L, Posewitz M, Schwarzer S, Happe T, et al. Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur J Biochem 2003;270:2750e8. [11] Ghirardi ML, King PW, Posewitz MC, Maness P e C, Fedorov A, Kim K, et al. Approaches to developing biological H2-producing organisms and processes. Biochem Soc Trans 2005;33:70e2. [12] Harris E. The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. San Diego: Academic Press; 1989.
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[13] Keller LR, Schloss JA, Silflow CD, Rosenbaum JL. Transcription of a-and b-tubulin genes in vitro in isolated Chlamydomonas reinhardtii nuclei. J Cell Biol 1984;98:1138e43. [14] Dekker J. The three ‘C’s of chromosome conformation capture: controls, controls, controls. Nat Methods 2006;3: 17e21. [15] Lewin B. Genes VII. Oxford: Oxford University Press; 2000. [16] Spilianakis CG, Lalioti MD, Town T, Lee GR, Flavell RA. Interchromosomal associations between alternatively expressed loci. Nature 2005;435:637e45.
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[17] Duan Z, Andronescu M, Schutz K, Mcllwain S, Kim YJ, Lee C, et al. A three-dimensional model of the yeast genome. Nature 2010;465:363e7. [18] Kalhor R, Tjong H, Jayathilaka H, Alber F, Chen L. Genome architectures revealed by tethered chromosome conformation capture and population-based modeling. Nat Biotechnol 2012;30:90e8. [19] Madigan MT, Martinko JM, Dunlap P, Clark DP. Brock biology of microorganisms. 11th ed. San Francisco: Benjamin Cummings; 2008.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii Elvira R. Eivazova, Sergei A. Markov* Biology Department, Austin Peay State University, P.O. Box 4718, Clarksville, TN 37041, USA
article info
abstract
Article history:
The direct relationship between hydrogenase gene conformation and its function in green
Received 23 July 2012
alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation
Received in revised form
in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of
6 September 2012
C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture
Accepted 13 September 2012
technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene
Available online 11 October 2012
region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the
Keywords:
gene region is present.
Biohydrogen
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
[FeFe]-hydrogenase gene
reserved.
Gene expression Chromosome conformation capture (3C) Chlamydomonas reinhardtii
1.
Introduction
Molecular hydrogen (H2) is one of the possible energy carriers of the future [1]. It is considered a fuel with low environmental impact since its combustion product (water) is non-polluting. Hydrogen is a renewable form of energy; it can be produced from water again. The conventional industrial methods for H2 production are costly and the problem has been to find a cheaper way to produce hydrogen. Biological H2 (biohydrogen) production by algal species has a number of advantages and it could be a cost effective alternative to the current industrial methods of H2 production [2]. Microscopic green algae produce H2 in photosynthetic reactions from water using sunlight as an energy source. Hydrogen production based on green algae holds the promise of generating
a renewable hydrogen fuel due to availability of large amounts of solar light and water. Green algae utilize the enzyme hydrogenase, which catalyzes H2 production, but only under anaerobic conditions, because the enzyme is very sensitive to O2 [3,4]. In addition, the hydrogenase gene is not expressed in the presence of O2 [5], but is rapidly expressed during anaerobic adaptation of the cells. Molecular mechanisms that regulate the hydrogenase gene expression under anaerobic conditions are poorly understood. Experimental evidence was provided recently that expression of a gene or gene family can be regulated through looping and bending of chromosomes [6e8]. Therefore, we expect that a hydrogenase gene region in green algae may have a spatial configuration that will have a considerable effect on gene activation, thus providing a mechanism for gene regulation.
* Corresponding author. Tel.: þ1 931 221 7440; fax: þ1 931 221 6323. E-mail addresses: [email protected] (E.R. Eivazova), [email protected] (S.A. Markov). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2012.09.087
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
Our hypothesis was that the hydrogenase gene in green algae adopts a specific conformation required for gene expression. Changes in such conformation provide the means for transcriptional activation of the hydrogenase genes under anaerobic conditions or their silencing under aerobic conditions. The gene conformation was assessed by the chromosome conformation capture (3C) assay, which is designed to estimate the interactions between genomic regions in vivo and to model a 3-D chromosome organization in the gene [9]. The basic principle of this method is that intact nuclei are isolated from cultured live cells and subjected to paraformaldehyde cross-linking to fix those segments of genomic DNA (via attached proteins) that are in close physical proximity to each other (Fig. 1). Cross-linked genomic DNA is digested with a restriction enzyme of choice and ligated at very low DNA concentrations that favor intramolecular interactions. Products of ligation are analyzed by PCR using primers designed for each restriction site to test for possible interactions between the restriction sites. The presence of a specific PCR product would indicate relative proximity of any two restriction sites to one another captured at a given point in time. We studied the microscopic eukaryotic organism, green alga Chlamydomonas reinhardtii. In order to implement 3C method, the whole genomic sequence of the experimental organism is required. A draft of the entire genome of Chlamydomonas reinhardtii CC-503 cw92 (mtþ) became available in 2004 and annotated in 2008. Green algae, along with some anaerobic bacteria and protozoa, possess [FeFe]-hydrogenases. The [FeFe]hydrogenase gene, hydA1 (referred to as HydA1 in some publications) from C. reinhardtii has been isolated and characterized [3]. A second [FeFe]-hydrogenase gene, hydA2, was identified in C. reinhardtii in 2003 [10]. It was reported that [FeFe]hydrogenase genes are transcriptionally regulated by presence or absence of O2 and by other unknown factors [11]. Therefore, a better understanding of the fundamental biology
17789
of hydrogenase gene (hydA1) expression would help the future development of economically viable systems for H2 production.
2.
Materials and methods
2.1.
Cell culture
The Chlamydomonas CC-503 cw92 (mtþ) cell wall-less strain was obtained from Dr. Elizabeth Harris (Chlamydomonas Genetics Center, Duke University). It was grown in a Triseacetateephosphate medium, pH 7.0, in 250-mL Erlenmeyer flasks with a cell suspension volume of 100 mL [12]. Continuous light was provided by cool white fluorescent lamps (15 mmol m2 s1 on the surface of the culture). Threeday-old cultures of green algae were used in experimental procedures. Cells were harvested at OD665 w 0.45 corresponding to a chlorophyll concentration of w17 mg ml1.
2.2.
Hydrogen production
Samples of cell suspension (3 mL) were vacuum degassed (270e300 torr) and flashed with N2 to induce anaerobiosis. Following this procedure, the samples were incubated under illumination provided by cool white fluorescent lamps (15 mmol m2 s1 on the surface of the culture) for 12 h in sealed 15 mL vials fitted with rubber stoppers. Hydrogen production was measured using a Gow-Mac gas chromatograph (Bethlehem, PA) equipped with a molecular sieve 5A column and a thermal conductivity detector. Nitrogen was used as a carrier gas.
2.3.
3C assay
We used the 3C assay [9] with some modifications.
2.3.1.
Nuclei isolation
The assay was performed with nuclei isolated from three-dayold algal cultures. To create anaerobic conditions, some of the cell cultures were additionally anaerobically incubated in sealed Erlenmeyer flasks under N2 atmosphere for 12 h in order to induce the expression of the hydrogenase gene. Nuclei were isolated by the modified method of Keller et al. [13]. Before nuclear isolation, cells were diflagellated by pH shock. A protease inhibitor was added (Pefabloc from Sigma, St. Louis, MO, 10 mL/mL), followed by 1% Triton X-100 (Sigma, St. Louis, MO) to lyse the cells. Nuclei were identified and counted in the lysed cell material using the fluorescent DNAbinding dye SYBR green from Molecular Probes, Eugene, OR (1:10,000 dilution) and a Nikon fluorescence microscope (Japan) equipped with the Nikon Digital Camera DMX1200F.
2.3.2. Fig. 1 e Schematic representation of the 3C methodology: formaldehyde-induced DNA attachment via protein, AatII restriction enzyme digestion, and PCR-mediated detection of ligation products after reversal of the cross-links (protein removal).
Formaldehyde cross-linking of proteins and DNA
Formaldehyde (Sigma St. Louis, MO) was added directly to isolated intact nuclei to a final concentration of 1% in a buffer to cross-link proteins and DNA. Nuclei were incubated and gently mixed for optimal cross-linking; after 10 min glycine (0.125 M final concentration) was added to stop the reaction. To remove non-cross-linked proteins from DNA, SDS (Sigma
17790
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 7 7 8 8 e1 7 7 9 3
St. Louis, MO) was added to a final concentration of 0.1% for 10 min, followed by Triton X-100 at concentration of 1%.
2.3.3.
Restriction digestion
Cross-linked DNA was digested with the AatII restriction enzyme (New England Biolabs, Beverly, MA). The restriction digestion was optimized by varying the concentration of enzymes and the reaction volumes. Digestion was most successful at a final volume of 50 mL with 3 mL of enzyme and 10 mL of DNA. The incubation time for digestion of chromatin was 1 h at 37 C. The mixture was incubated with 1.6% SDS for 20 min at 65 C to inactivate AatII.
2.3.4.
Ligation
To favor intermolecular interactions the DNA material was ligated at a low DNA concentration. We established optimal conditions for ligation (T4 ligase, New England Biolabs) by using DNA at different concentrations and monitoring products of ligation by PCR. DNA was ligated for 1 h at 16 C. After ligation, the mixture was incubated with 1.6% SDS for 20 min at 65 C to inactivate AatII.
2.3.5.
Reverse cross-linking
Cross-linking of proteins and DNA was reversed by overnight incubation of the samples with proteinase K (Promega, Madison, WI) at 65 C.
2.3.6.
DNA purification
five restrictions sites. Primer pairs homologous (1þ1, 2þ2, 3þ3, 4þ4, 5þ5) that do not showed any PCR products were eliminated from consideration (from our further experiments). For example, we had primers B5 and B6 and B7 and B8 for our restriction site 1 (Fig. 2). Primers B7 and B8 did not show any PCR products and were never used again. Forward and reverse primers for PCR amplification were used in all pairwise homologous (1þ1, 2þ2, 3þ3, 4þ4, 5þ5) and nonhomologous (1þ2, 2þ1, 1þ3, 3þ1, 1þ4, 4þ1, 1þ5, 5þ1, etc.) combinations. In other words, every forward primer for one restriction site was combined in pairs with a reverse primer of every other restriction site in order to complete the set of all possible primer pair combinations. The PCR products were resolved by electrophoresis on 2% agarose gels.
3.
Results
3.1. Hydrogen production as a measure of hydA1 gene expression To find out when hydrogenase gene is expressed, we measured H2 production by C. reinhardtii cells during several days. Three- and four-day-old cultures showed significant levels of H2 production under anaerobic conditions (Fig. 3). We did not detect any H2 production under aerobic conditions. The three-day-old cultures with the maximal H2 production were used in the gene conformation experiments (3C assay).
DNA was extracted by standard “phenolechloroform” method.
3.2. 2.3.7. Analysis of interactions between restriction sites in hydrogenase gene region Primer pairs were designed (and synthesized by Sigma Genosys, Woodlands, TX) for each of the five AatII restriction sites (Fig. 2) positioned along the 29-kb hydrogenase gene region to make approximately 250 bp long DNA products. Originally, two pairs of primers were designed for each of the 159990 bp
130396 bp Base position Chlamydomonas hydA1
Search for the gene (hydA) on C. reinhardtii DNA
To perform the 3C assay (DNA conformation assay) the hydA1 gene location on C. reinhardtii DNA must be known. Using the database from the Joint Genome Institute (http://www.jgi.doe. gov) and sequences of a hydrogenase gene and its promoter from Genbank (accession numbers AJ012098 and AJ308413), the whole genome sequence of C. reinhardtii has been searched. The [FeFe]-hydrogenase gene (hydA1) region (29 kb) has been located in contigs 20 and 21 (scaffold 12) within a base position range of 130396 and 159990 (Fig. 2). The position of the promoter was located manually using the Chlamydomonas whole DNA sequence and hydA1 data deposited in Genbank (accession numbers above) by Happe and Kaminski [3].
AatII sites 1
2
3
4
5
Fig. 2 e Schematic representation of the 29-bp hydA1 (gene) region on Chlamydomonas chromosome. Locations of AatII restriction sites are shown. Base count is indicated according to the US Department of Energy Joint Genome Institute website (http://www.jgi.doe.gov). Positions of the five restriction sites from the start of the hydA1 promoter as follows: site 1, L10206; site 2, L7049; site 3, L2136; site 4, D498; site 5, D15055. The bolded area in a Chlamydomonas hydA1 gene indicates the promoter region.
Fig. 3 e Hydrogen production by Chlamydomonas reinhardtii as a function of time. A e H2 production under anaerobic conditions; - e H2 production under aerobic conditions.
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3.3.
Search for the restriction enzyme
The gene conformation 3C methodology required to perform DNA restriction enzyme digestion (Fig. 1). The specific restriction enzyme is needed which can cut C. reinhardtii DNA several times in hydA1 gene region and, at least one time, in directly within gene promoter [7,9]. DNA fragments after restriction digestion should be around 3000 bp in size. Using the Webcutter2 (http://rna.lundberg.gu.se/cutter2) we found a restriction enzyme, AatII, which produced five restriction sites in the hydA1 gene region of C. reinhardtii DNA (Fig. 2) along the 29 kb gene region. Several other enzymes were considered for our experiments such as NotI. Compared to AatI, NotI can make only 3 cuts in the 29 kilobase (kb) chromosome region of our interest.
3.4.
Application of 3C methodology
3.4.1.
Nuclei isolation
The first step in 3C assay was to isolate nuclei. The method employed for nuclei isolation allowed us to visualize in real time the release of nuclei from algal cells treated with detergent (Fig. 4). Isolated nuclei were uniform in size and their appearance was similar to nuclei of intact cells.
3.4.2. Formaldehyde cross-linking of proteins and DNA, restriction digestion and DNA ligation Isolated nuclei were subjected to formaldehyde treatment to fix DNA sites that are in close physical proximity to each other. Cross-linked DNA was digested with AatII restriction enzyme and DNA fragments were ligated at very low DNA concentrations that favor intermolecular interactions.
3.5. Specific interaction between restriction enzyme sites: gene conformation under aerobic and anaerobic conditions Availability of cross-linked DNA ligation products were assessed using PCR using primers to AatII restriction sites in all possible combinations. As a control, we digested non-crosslinked DNA (not subject to formaldehyde treatment) with restriction enzyme AatII followed by ligation and PCR. We
Fig. 4 e Nuclei pop out of Chlamydomonas cells after their treatment with 1% Triton X-100. Picture taken using Nikon fluorescence microscope equipped with the Nikon Digital Camera.
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followed the general control guidelines described by Dekker [14]. Representative PCR products with homologous primers (5þ5) and non-homologous primers (2þ5, 3þ5) from crosslinked DNA of aerobically grown algae are shown in Fig. 5A. The obtained experimental results allow us to conclude that restriction site 5 interacts with sites 2 and 3. Because of the presence of PCR products obtained with the primers specific for restriction sites, located several kilobase (kb) away from each other, we reason that these sites are positioned in close proximity by looping of chromosome. Thus, these data provided important evidence for a loop configuration in the hydrogenase gene region under aerobic conditions (Fig. 6A). In DNA templates obtained under anaerobic conditions we did not observe any interactions between the restriction sites, thus there was no any loop organization in the hydrogenase gene region (Figs. 5 and 6B).
4.
Discussion
Gene expression is regulated at different levels in the pathway from gene to functional protein. For most genes the initiation of transcription is the most important point of control [15]. Our understanding of gene regulation is moving from a one-dimensional picture of regulatory DNA sequences (“sending” proteins to express or silence genes) into a threedimensional spatial organization of chromosomes influencing gene expression [16e18]. Physical looping of DNA with the help of proteins permits gene expression or silencing in eukaryotes and, possibly, in prokaryotic microorganisms [19]. With the introduction of chromosome conformation capture (3C) methodology [9] it has now become possible to study the role of chromosome conformation in the course of gene expression or silencing. This method gives a new dimension to the study of gene regulation by determining the cross-linking frequencies of interaction between different chromosome segments within a gene region in vivo, and to model chromosome conformation before and after gene expression. This approach was successfully applied in a mammalian system to interferon gamma gene, Ifng [7]. The chromatin conformation of the Ifng gene was analyzed in T cell. The results showed a looping structure, which changed upon the Ifng expression during T cell differentiation. In such multi-looped conformation, several distal regions were brought into close proximity to the promoter region, forming a so-called “chromatin hub” configuration. We have analyzed chromosome conformation in the 29 kilobase (kb) region containing [FeFe]-hydrogenase gene
Fig. 5 e A & B. Representative PCR products generated with cross-linked DNA templates of C. reinhardtii cultured aerobically (A) and anaerobically (B).
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regulation of gene expression. The results showed a loop organization within the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. We reasoned that because DNA is folded within the hydrogenase gene region the enzyme RNA-polymerase cannot transcribe the [FeFe]-hydrogenase gene. In contrast, under anaerobic conditions, when the [FeFe]-hydrogenase gene is active (expressed), no loop conformation in the gene region was found. This study also identified functionally significant fragments in the hydrogenase gene (hydA1) region. In future work, these fragments could be selectively deleted in order to express the hydrogenase gene, even in the presence of molecular oxygen.
Acknowledgments This work was supported by National Science Foundation (USA), award # 0821571 and Austin Peay State University and DePauw Universities Summer Faculty Research Programs. We thank Ms. Brittany Durr for valuable assistance with the experimental work.
Fig. 6 e [FeFe]-hydrogenase gene region of a green alga Chlamydomonas reinhardtii under aerobic (A) and anaerobic conditions (B).
(hydA1) of C. reinhardtii by 3C technique under aerobic and anaerobic conditions. The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions (Fig. 6A). In this loop conformation, parts of the chromosome around the hydrogenase gene were brought into close spatial proximity to each other, thus effectively silencing this gene. Chromosome conformation of the hydrogenase gene region changed to “unfold configuration” (Fig. 6B) upon transition of cells to anaerobic conditions which provided the means for transcriptional activation of the hydrogenase gene and subsequent H2 production. The observed correlation between hydA1 gene activity and its conformation implies the regulatory mechanism for hydrogenize gene activation and silencing by the sequential changes in its spatial configuration. Our data are important to advancing knowledge and understanding of hydrogenase gene expression, because it presented for the first time the experimental evidence of the conformational regulation of such gene expression in algae. Better understanding of the regulation of hydrogenase genes will help to optimize H2 production by microscopic algae and other microorganisms. New strains of algae that have highly expressed hydrogenase genes could be engineered. This research is also contributed to our general understanding of mechanisms of gene regulation in living organisms.
5.
Conclusions
The data presented in this paper for the first time showed the mechanism of [FeFe]-hydrogenase gene (hydA1) expression in the green alga C. reinhardtii, or as we called it, the conformational
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