Identification of scaffold/Matrix Attachment (S/MAR) like DNA element from the gastrointestinal protozoan parasite Giardia lamblia
© Padmaja et al; licensee BioMed Central Ltd. 2010
Received: 23 December 2009
Accepted: 18 June 2010
Published: 18 June 2010
Chromatin in the nucleus of all eukaryotes is organized into a system of loops and domains. These loops remain fastened at their bases to the fundamental framework of the nucleus, the matrix or the scaffold. The DNA sequences which anchor the bases of the chromatin loops to the matrix are known as Scaffold/Matrix Attachment Regions or S/MARs. Though S/MARs have been studied in yeast and higher eukaryotes and they have been found to be associated with gene organization and regulation of gene expression, they have not been reported in protists like Giardia. Several tools have been discovered and formulated to predict S/MARs from a genome of a higher eukaryote which take into account a number of features. However, the lack of a definitive consensus sequence in S/MARs and the randomness of the protozoan genome in general, make it a challenge to predict and identify such sequences from protists.
Here, we have analysed the Giardia genome for the probable S/MARs predicted by the available computational tools; and then shown these sequences to be physically associated with the nuclear matrix. Our study also reflects that while no single computational tool is competent to predict such complex elements from protist genomes, a combination of tools followed by experimental verification is the only way to confirm the presence of these elements from these organisms.
This is the first report of S/MAR elements from the protozoan parasite Giardia lamblia. This initial work is expected to lay a framework for future studies relating to genome organization as well as gene regulatory elements in this parasite.
Sequencing and annotation of the different genomes done in the last couple of decades has clearly shown that even the relatively compact eukaryotic genomes have large amounts of non-coding DNA. This DNA harbors elements that control genomic activity such as gene regulators, non-coding RNAs and less well characterized elements that position the chromosomes on the nuclear matrix. The nuclear matrix forms a three dimensional protein network onto which chromatin fibers are attached. Interaction between chromatin and the nuclear matrix is believed to occur at specific sites from 300 base pairs (bp) to several kilobases (kb) long, termed scaffold/matrix attachment regions (S/MAR) .
Experimentally, SMARs have been defined as either DNA fragments that remain bound to the nuclear matrix after chromatin proteins and other DNA are removed, or DNA that binds to extracted nuclear matrix in the presence of competitor DNA [2, 3]. Identification of S/MARs is a necessary step for successful functional mapping of nucleotide sequences, since these sites can bring genes into association with the nuclear matrix and significantly change transcription level, thus marking transcriptionally active regions . S/MAR elements play a major role in genome organization and gene regulation. They have been reported to alter the expression levels of some genes depending on their position relative to the matrix . S/MARs have also been associated with enhanced transcription, particularly in transgene constructs where flanking transgenes with S/MARs have resulted in higher and more stable expression . They have been associated as a boundary between functional chromatin domains [7, 8]. It is also reported that the effects of long-range enhancers may be restricted by the positioning of S/MAR elements . From the genome organization perspective, S/MARs have been implicated in the positioning of chromosomal territories [7, 10].
Computational methods are thought to be prerequisite for the analysis of whole genomes for predicting S/MARs and though several tools like MarWiz [11–13], Marscan , ChrClass , SMARtest  and SIDD [16, 17] have been developed for this purpose, prediction of S/MAR is not conclusive unless it has been supported by experimental proof. The most common experimental method for identifying S/MAR uses re-association assays to define DNA fragments that bind to the nuclear matrix . South-western assays [19, 20] and PCR based assays  have also been used successfully to show S/MAR binding to nuclear matrix.
Though S/MARs have been well studied in yeasts, plants, mammalian systems and Drosophila, there has been very few reports of these elements from the protists. So far genome wide search for S/MARs have been carried out in silico for Arabidopsis thaliana and C. elegans using SMARTest and MRS finder respectively [22, 23]. This study had revealed that genes containing predicted S/MARs had low transcription levels . In C.elegans, S/MARs were found to be the flanking coding regions . Marfinder and Marscan have been used previously to identify functional S/MAR elements in Entamoeba.
The genome of Giardia lamblia, the protozoan parasite responsible for causing Giardiasis worldwide among people with poor fecal-oral hygiene, has been sequenced recently . The 11.7 Mb genome of this deep branching eukaryote, distributed over 5 chromosomes showed an exceedingly simple genome structure comprising of only 2 origin recognition complex proteins and total absence of regulatory initiation proteins . Moreover, Giardia contained only 4 of the 12 transcription initiation factors present in Saccharomyces. As the genome of this organism has been studied, very few regulatory elements were seen to be present in this parasite. Promoters had been identified and characterized earlier [26–32] but other regulatory elements like insulators, boundary elements enhancers and locus control regions were not revealed in the genome sequencing project.
In this work, we have used all the available bioinformatics tools for predicting S/MARs from the genome of Giardia lamblia and used PCR based, as well as south-western assays to actually see how many of the predicted S/MARs were able to bind to nuclear matrix. This is the first ever report of S/MAR like DNA elements from this gastrointestinal pathogen. In this paper we have also reflected on how any single computational tools for prediction of S/MAR can be very inaccurate on the protozoan parasite genome, but a combination of different tools along with laboratory based assays, give us a comprehensive idea about S/MAR distribution in Giardia lamblia genome. Our studies show 10 S/MAR sequences from Giardia lamblia which are associated with its nuclear matrix proteins are can thus be regarded as S/MAR elements.
In silico prediction of putative S/MARs from Giardia genome using existing tools
Summary from various S/MAR identification tools for Giardia
# of S/MARs identified
Average length of identified S/MARs (bp)
Av distance between S/MARs (bp)
Too less prediction for calculating loop size.
Summary of nuclear matrix binding ability of predicted Giardia S/MARs
Binding to Giardia
By PCR Based Assay
By South Western Assay
Organization of predicted S/MARs in Giardia
Of the 15 putative S/MARs, some were found to have some interesting organization. GlSMAR7 (Figure 2B) was found to have ORF of a reverse transcriptase endonuclease apart from a VSP and High Cysteine protein within 12 kb of it. Similar organization i.e presence of several reverse transcriptase endonuclease was also noticed in GlSMAR22 (Figure 2B) These ORFs are reported to be present in the telomeric region of the chromosome in Giardia. The significance of the presence of these elements in close proximity of such ORFs is beyond the scope of current study. The 15 putative S/MARs were then tested experimentally for their ability to bind to nuclear matrix. The results are summarized in Table 2.
Giardia S/MAR like elements are present in the nuclear matrix
Giardia S/MARs bind to nuclear matrix proteins
Mass Spectrometry of Giardia nuclear matrix protein
To verify whether any of the proteins recognized by the S/MARs from Giardia were indeed resident nuclear matrix proteins, we excised one band (44 kd) from the coomassie stained gel which also bound to GlSMAR7 (Figure 4B a) and went for mass spectrometric identification.
Giardia lamblia, is a diplomonad, with 2 nuclei and is often referred to as a "deep branching eukaryote" as it diverged out of the main evolutionary tree long before the other eukaryotes. As a result, this oraganism has a number of unique features which have become more "organized" in the higher eukaryotes. One of the most unique features of Giardia is its lack of organellar structures as for example a well defined mitochondria, Golgi bodies and endoplasmic reticulum, in spite of being an eukaryote. Traces of marker proteins from these organelles and an amazingly developed membrane structure adept to carry out these functions are however present here enabling this organism to be classified as a eukaryote .
Though the initials reports of nuclear matrix go as far back as 1960's, the research on S/MARs as potential regulatory elements come from the works of J. Bode in 1988 [40, 41]. Since then, throughout the eukaryotic world, the S/MARs have been found to play a significant role in the organization of chromatin, and gene regulation. Studies on the recently sequenced Giardia genome have shown the genome to be unique in its own way. The protist has 5 chromosomes, and almost 9000 ORFs packed into a small genome of 12 Mb length. It has been seen that the parasite has no homolog for H1 which is the universal linker for compacting chromatin in the nuclei . In this scenario, the study of Scaffold/Matrix attachment region in this parasite can shed adequate light on the chromatin organization in this organism. We did a preliminary screen on the G.lamblia genome with available S/MAR prediction tools. When the common regions predicted by at least 2 tools were taken into consideration, we were able to shortlist at least 15 putative S/MAR regions. To prove this DNA fragments were indeed nuclear matrix dependent fragments we did the PCR based assay, which showed that 10 out of 15 putative S/MARs where actually associated with the nuclear matrix of Giardia. This showed that the false positive rate of our strategy was about 33%. Assuming that the distance between the S/MARs in this genome can range from 50-160 kb, as seen in Table 1, we expect about 110 S/MARs in the entire genome of Giardia. The combination of computational tools correctly predicted only 10% of the total number of the expected S/MARs. This indicates that the S/MAR prediction tools that can be used with accuracy on the higher eukaryotic genome, in most of the instances are not very accurate in predicting lower eukaryotic S/MARs. Experimental methods are an absolute necessity in correctly identifying these elements from the lower eukaryotic genomes. The computational tools for S/MAR predictions can only be used as an initial screen for scanning the genome of the protists for presence or absence of S/MARs, but the actual confirmation is achieved only by experimental methods. Of the 10 S/MARs, 8 also showed positive nuclear matrix binding property in south western blots. Among these, 7 S/MARs which showed positive binding both in PCR as well as south western assay were indeed true S/MARs. It now remains to test these Giardia S/MARs for chromosomal organization studies.
One of the major properties of S/MARs is chromosome organization, anchoring and maintenance of higher order structure . This is achieved by the proteins in the nuclear matrix which bind to the S/MARs thereby allowing it to carry out these functions. The proteins in the nuclear matrix are involved in a host of different functions, including DNA replication and repair . Of these the S/MAR binding proteins are (S/MARBP) are of utmost importance as they regulate transcription, replication, repair and regulation of gene expression . One of the GlSMARs, GlSMAR7 bound to a proteasome subunit 8 as shown by our mass spectrometry results. The 26 S proteasome is an eukaryotic ATP-dependent protease complex of 2000 kd which is reported to be present in the nuclear matrix in mouse myoblasts . As seen in Figure 5, the conserved domain in the 26 S proteasome subunit 8 in Giardia was a AAA domain belonging to the ATPase binding protein superfamily. These proteins perform a diverse range of functions in the cell starting vesicle fusion, peroxidase biogenesis  to DNA repair . Thus it is not unlikely that this protein would be associated with S/MARs and have DNA binding properties. There have been reports on the proteasome 20 S of Giardia lamblia[47, 48], where Emmerlich et al showed the 14 subunits making up this proteasome structure. Though the annotated genome of Giardia shows the presence of several of the proteasome 26 S subunits, no detailed analysis has been done on these proteins in Giardia. A detailed phylogenetic analysis of another AAA ATPase domain containing protein Midasin has been studied by Gallego et al. . This protein is conserved thoughout eukaryotes and plays the role of a nuclear chaperone in most organisms. One of the proteins found to be associated with S/MARs from yeast to humans, is the SAF Box domain containing protein. As reported by Kipp et al in 2000 , SAF-A binds to S/MARs through a novel conserved protein domain. A search in http://www.eupathDB.org for proteins having the SAF box or the SAP domain showed that 47 such proteins were present in the different protozoan genomes (Cryptosporidium, Plasmodium, Toxoplasma, Entamoeba and Trichomonas). Thus it is likely that these genomes will also have S/MAR like elements in their genome. However, when searched in the Giardia genome, this SAF/SAP domain containing protein was not present. Our experimental results discussed in this work indicate that Giardia has S/MAR binding protein (26 S proteasome subunit 8) which does not have a SAF/SAP domain, but has nucleotide binding domains. While it is possible that in a recently sequenced genome, this protein was not annotated, it is also possible that Giardia placed much earlier in the evolutionary scale probably has not yet defined a machinery where these highly conserved domain containing proteins may be present. The presence of S/MARs in Giardia and the absence of SAF box proteins in this organism may also indicate that the early divergence of Giardia during evolution probably .resulted in "missing out" this very conserved protein involved in nuclear architecture.
S/MARs have been found to be associated with not only chromatin anchoring but also with other regions of the genome as introns  and can play a significant role in the regulation of gene expression [52, 53]. Studies on S/MAR in Arabidopsis and maize [54, 55] have shown that the plant genome is not packaged by random gathering into domains of indiscriminate length, but rather, the genome is gathered into specific domains, and a gene consistently occupies a discrete physical section of the genome. The average loop size in Arabidopsis and maize has been estimated as 25 and 45 kb, respectively , though other studies  have suggested smaller domain sizes. Some loops may remain permanently condensed and inactive, even within the euchromatic portions of the genome, whereas others can be extended to produce a transcriptionally poised conformation in appropriately differentiated cells . Our analysis for a genome-wide distribution of S/MARs using different tools indicates that the loop size ranges from 50-160 kbp in Giardia (Table 1). Data on the location of transcribed elements within structural loops at the supragenic level suggest that attachment to the matrix and transcription is not systematically associated [57, 58], though S/MARs are associated with the ends of some DNaseI-sensitive (transcriptionally poised) domains . S/MARs have also been identified within introns of genes [60, 61]. Cockerill et al.  suggested that S/MARs flanking enhancer sequences may act as positive and/or negative regulators of enhancer function. It is presumed that additional specific S/MARs have been further demonstrated in a variety of functional tests to act as insulators , according to the loop domain model, by protecting a loop from the effects of the neighboring chromatin or associated enhancer sequences. Distribution of Giardia S/MARs among the transcription factors also hints at this possibility (data not shown). A much more in-depth study of the S/MARs in lower eukaryotes is required to understand the chromatin dynamics and packing in these organisms.
An observation was made in the study by Linnemann et al in 2009, where it was seen that the S/MARs when present in the 5' region of a gene resulted in a transcript presence, where as those present within the ORF associated with silenced genes. A number of S/MARs in Giardia were also found within the ORFs. The significance of this is not clear. In Entamoeba, such S/MARs were found to have reduced binding ability to nuclear matrix compared to the ones that were present outside ORF (our unpublished data). It is possible that in these early eukaryotes, the genome organization machinery is also in early stages of evolution and the S/MARs within the ORFs are actually the ones which in course of evolution would lose their ability to bind to the nuclear matrix completely.
Though analysis of S/MAR on large genomic sequences are being done [63–65], S/MAR regions of protists have never really come to the limelight. Study on the S/MARs in these organisms is of significance in the understanding their gene organization and regulation. The multiple roles played by these S/MARs starting from chromosome organization to promoter control, acting as domain barriers, make them important regulatory elements which have not received much focus yet. Most of the prediction tools are designed with the structurally organized higher eukaryotic genome in mind. A comparison of these tools reveal that no single tool is accurate enough to predict the S/MARs even from an organism with a well defined genome structure . In case of lower eukaryotes, these tools do identify the S/MARs, but with much less accuracy. Our study clearly indicated that even if we take into consideration all the available tools for predicting S/MARs from protozoan parasites, they have to be verified experimentally for their ability to be associated with the nuclear matrix. Studies like this, also indicate the need to modify and develop more dedicated tools for the prediction of these elements from such divergent genomes, which in turn would help to study gene organization and gene regulation in a much wider scale in these protozoans.
Bioinformatic tools for prediction of S/MARs
G. lamblia genome sequences available at http://www.Giardiadb.org were used for all analysis. S/MAR analysis was done according to the available S/MAR analysis tools - Marfinder (downloaded from http://genomecluster.secs.oakland.edu/marwiz/) SMARTEST (http://www.genomatix.de/smartest.html), Marscan (EMBOSS) and Chrclass (http://ftp.bionet.nsc.ru/pub/biology/chrclass/chrclas2.zip). The tools and the description of the datasets as well as the parameter information were done according to Evans et al, with modifications wherever required.
Giardia cell culture
Giardia lamblia strain WB1267 was cultured axenically in TYI-S media supplemented with 10% Adult Bovine serum (Invitrogen) and 1 mg/ml of bovine bile (Sigma) in 50 ml culture flasks. Parasites were routinely subcultured every 48-72 hours when confluent. Cells were harvested for nuclear matrix isolation by chilling on ice for 20 mins followed by harvesting at 2000 rpm for 5 min in extraction buffer (10 mM Hepes, pH 6.8; 24 mM KCl; 10 mM MgCl2) in the presence of protease inhibitor cocktail (Sigma, USA).
Genomic DNA, Designing primers and PCR
Giardia lamblia (strain WB1267) genomic DNA was prepared from confluent Giardia cultures using the Genomic DNA Isolation kit (Sigma) according to the manufacturers instruction. Primers were designed against the predicted S/MAR sequences (details in additional file 1; Table S1) and supplied by Ocimum Biosolutions, India. Putative S/MARs were amplified from the G. lamblia genome by Polymerase Chain Reaction using Taq polymerase (NEB, USA).2 ng G.lamblia genomic DNA was used as a template. DNA was denatured at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s and extension at 72°Cfor 30 s. Final extension of 72°C was kept for 7 min. Amplicons were sequenced to confirm the genomic sequence.
Synthesis of probes for hybridization
The purified PCR products were used as templates for the labeling reaction. Biotinylated dNTPs (NEB) and NEBlot Kit (NEB) was used to label the probes for chemiluminiscent detection. The reaction for synthesis of probes was done according to the manufacturer's instruction.
Isolation of nuclear matrix
1. Cultured G. lamblia cells were harvested at 2000 rpm and washed once in phosphate buffered saline (PBS). The cells were lysed in Extraction buffer (10 mM HEPES-KOH(pH 7.2), 24 mM KCl, 10 mM MgCl, 1 mM E64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane), a protease inhibitor; 1 mM, PMSF, 2 mM DTT, 0.03%NP-40). The lysate was loaded on a cushion of Extraction buffer containing 0.8 M sucrose and centrifuged at 6000 rpm for 20 minutes. The nuclei were recovered in the pellet.
Nuclear matrices were prepared by treatment of the isolated nuclei with 50 U of DNase I at 37°C for 30 minutes and centrifuged at 6000 rpm for 10 minutes . The pellet was then washed twice with Low Salt Buffer (LSB) containing 10 mM HEPES-KOH(pH 7.2), 0.2 mM MgCl2, 10 mM 2-mercaptoethanol followed by treatment with High Salt Buffer (HSB) containing 1.6 M NaCl, 10 mM HEPES-KOH(pH 7.2), 0.2 mM MgCl2,10 mM 2-mercaptoethanol and incubated at 4°C for 15 minutes. The insoluble nuclear matrix proteins were separated from the high salt extractable proteins by centrifugation at 6000 rpm for 10 minutes. The final pellet was further washed with 0.5% Triton-X 100. All fractions were prepared in SDS-PAGE gel loading buffer and separated on a 10% SDS-PAGE.
PCR-based assay for S/MAR binding
Nuclear matrix was isolated from G.lamblia cells as described above. For the PCR based assay the protocol of Kramer  was used with modifications. Briefly, Giardia nuclei were washed once with LSB and then treated with HSB and incubated on ice for 15 min. Following incubation, the reaction mix was centrifuged at 6000 rpm and the supernatant was removed. The pellet was washed once again with LSB followed by 1 × restriction enzyme buffer for Eco R1. The pellet was then digested with Eco R1 for 2 hrs at 37°C. Following digestion, the reaction tube was centrifuged at 6000 rpm for 10 min and the supernatant was collected in a fresh tube. The residual pellet (nuclear matrix) and the supernatant were subjected to phenol: chloroform (1:1; v/v) treatment and the extracted DNA from both fractions were precipitated with equal volume of isopropanol. 2 ng of the extracted DNA was next used as a template for PCR for the different predicted S/MAR sequences in Giardia.
South western hybridization for detecting nuclear matrix-S/MAR association
The protein fractions separated by SDS-PAGE were transferred to PVDF membrane. The membrane was blocked with 3% non-fat skimmed milk in containing 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA (Standard Binding Buffer; SBB) for 2 hours. After three washes of 15 minutes with SBB the membrane was incubated overnight at 4°C with the biotinylated DNA probes. Unbound probe was washed with SBB followed by incubation with Streptavidin conjugated to Horseradish Peroxidase (Sigma 1:500 dilution) for 1 hour. Excess Streptavidin -HRP was washed with the same buffer. The DNA binding ability was then detected with an enzyme catalyzed light emitting reaction using Super Signal West Pico Chemiluminescent substrate kit (Pierce 34082) according to the manufacturer's instruction. The membrane was then exposed to CL-Xposure films (Pierce 34092) and the emitted light was captured on the film.
Sample preparation for proteomic analysis of Giardia nuclear matrix protein
A major band around 44 kDa was excised from the gel and sent to Syngene International, Bangalore, India for proteomic analysis.
The sample processing was done by the CRO according to standard methods. Briefly, the gel bands supplied were washed with water and chopped into ~1 mm cubes and washed with 50 mM NH4HCO3 and acetonitrile mixture (1:1) for 15 min and washing solution was aspirated completely. Sufficient acetonitrile was added to cover the gel particles following above the washing step. Acetonitrile was removed after 2 min and gel pieces were re-hydrated in 50 mM NH4HCO3. After 5 min, an equal volume of acetonitrile was added and incubated for 15 min followed by complete removal of all solvents. Gel pieces were covered by enough acetonitrile to effect shrinking of gel pieces. Following shrinkage of gel pieces, acetonitrile was removed and gel particles were dried in a vacuum centrifuge. For reduction and alkylation, the gel particles were allowed to swell in 50 mM NH4HCO3, 10 mM dithiothreitol (DTT) and incubated for 45 min at 56°C in a water bath followed by cooling to room temperature. Excess liquid was removed and replaced with freshly prepared 50 mM iodoacetamide in 50 mM NH4HCO3 followed by incubation for 30 min at room temperature in the dark. Excess iodoacetamide solution was removed and gel particles were washed twice with 50 mM NH4HCO3 and acetonitrile mixture (1:1). Each washing was carried out for 15 min. Gel pieces were allowed to dehydrate in acetonitrile followed by vacuum drying. For in-gel digestion, gel pieces were rehydrated in 20 ng/ul Trypsin (Sigma) solution prepared in 25 mM NH4HCO3 at 37°C for 30 min.25 mM NH4HCO3 was added to the reaction mixture so that the gels remained completely submerged. Digestion was allowed to proceed at 37°C for 16 h.
Following digestion, the peptides were extracted by adding extraction buffer (50% acetonitrile containing 0.1% TFA) to cover the gel pieces followed by sonication. The extract was collected after centrifugation and concentrated to a final volume of 10μl using vacuum centrifuge.
MALDI matrix preparation and MALDI-MS Analysis
Saturated solution of Alpha-cayano-4-hydroxy cinnamic acid was prepared using 30% acetonitrile containing 0.1%TFA to prepare the matrix for MALDI. Undissolved matrix particles were removed by centrifugation. Equal amount of sample and matrix were mixed in a microfuge tube and spotted on MALDI-target plate and the mixture was allowed to dry at room temperature.
MALDI spectra were acquired in an AUTOFLEX III SMARTBEAM MALDI-MS instrument (Bruker Daltonics, Germany). External calibration was done with peptide calibration standard supplied by Bruker, with masses ranging from 1046 Da-3147 Da. All the spectra were acquired in Reflectron +ve ion mode with an average of 2000 laser shots. Mass detection range was set between m/z 800-3500. Acquisition software used was FlexControl version 3 and Analysis software used was FlexAnlaysis version3. Analysis of the peaklist obtained was done using the web based analysis software MASCOT using the NCBInr database. All the default parameters of MASCOT were maintained for analysis.
Polymerase Chain Reaction
Scaffold/Matrix Attachment Regions.
This work was supported by the start-up fund obtained by AU-KBC Research Center, Chennai, India and Dept. of Biotechnology, Govt. of India. SP was supported by the Council for Scientific and Industrial Research, Govt. of India.
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