Name: associated chromosome trap for identifying long-range dna interactions
Uploaded: 2011-04-23T14:00:00
Duration: 14 min 49 s
Description: Watch this Scientific Journal Video about Associated Chromosome Trap for Identifying Long-range DNA Interactions at JoVE.com

We thanks Adelle Murell and Wolf Reik very much for sharing their 3C protocol. This work was supported by the Department of Defense and the Research Service of the Department of Veterans Affairs.

1. Formaldehyde fixation of long-range chromatin interactions
<ol>
	<li>Culture human cell line HL-60 in RPMI1640 medium with 15% FBS and 1 x penicillin/streptomycin to 80-90% confluence in an incubator supplied with 5% CO2 at 37&deg;C.</li>
	<li>Collect the cells into a 50-ml Nunc tube, centrifuge at 1,200 rpm for 15 minutes, and then remove the medium by aspiration</li>
	<li>Add 5 ml of culture medium to resuspend the cell pellets, and count the cells using a hemocytometer. Take approximately 1 x 107 cells to a volume of 40 ml with RPMI1640 /10% FBS, then add 1.7 ml of 37% formaldehyde to fix the dilute chromatin.</li>
	<li>Incubate at room temperature for 10 minutes with gentle shaking, and then quench with 2.4 ml of 2M glycine. Centrifuge for 15 minutes at 1,200 rpm at 4&deg;C, and remove the supernatant. Wash the pellet once with 40 ml ice cold PBS, then spin down the pellet and remove the PBS.</li>
</ol>
2. Cell lysis to isolate nuclei
<ol>
	<li>Resuspend cells in 40 ml of ice cold lysis buffer (10 mM Tris-HCl, pH8.0, 10 mM NaCl, 0.2% NP-40) with freshly added protease inhibitors (1:500 dilution) and 0.1 mM PMSF. Incubate in a cold room with rotation for 90 minutes, centrifuge at 2,500 rpm for 15 minutes, and remove the supernatant.</li>
</ol>
3. Restriction enzyme digestion with Bgl II
<ol>
	<li>Resuspend the nuclei in 0.5 ml of 1 x NEB buffer 3, and add 15 &mu;lof 10% SDS. Incubate at 37&deg;C for 1 hour with shaking, then add 45 &mu;l of 20% Triton X-100 to sequester the SDS. Incubate at 37&deg;C for 1 hour with shaking.</li>
	<li>Use an aliquot of 1 x 106 nuclei (about 15 &mu;g, one tenth of the original cells) for restriction enzyme digestion. Take out 55 &mu;l of nuclei solution from Step 3.1 and make up to 500 &mu;l with 433 &mu;l of 1 x NEB buffer 3 and 12 &mu;l of Bgl II(50U/ul). Incubate at 37&deg;C overnight.</li>
</ol>
4. Ligation of interacting DNA segments
<ol>
	<li>Inactivate the restriction enzyme by adding 95 &mu;l of 10% SDS, and denature by heating at 65 &deg;C for 20 minutes in a water bath.</li>
	<li>Add 7 ml of 1 x ligation buffer (30 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM DTT, 1 mM ATP) and 360 &mu;l of 20% Triton X-100 and incubate at 37&deg;C for 1 hour.</li>
	<li>Lower the temperature to 16&deg;C and add 50 &mu;l of 400 U/&mu;l T4 DNA ligase. Incubate the sample at 16 &deg;C for 4 hours and then at room temperature for 30 minutes.</li>
</ol>
5. DNA Purification
<ol>
	<li>Add 300 &mu;g of proteinase K, and incubate at 65&deg;C overnight.</li>
	<li>Add 5 &mu;g of RNase A and incubate at 37&deg;C for 30 minutes</li>
	<li>Purify DNA by phenol/chloroform extraction, and precipitate DNA in isopropanol. Dissolve DNA in 150 &mu;l of sterile distilled water.</li>
</ol>
6. Digestion with Msp I and ligation with oligonucleotide linkers
<ol>
	<li>Incubate 2&mu;g of purified DNA with 5 units of Msp I at 37&deg;C for 4-6 hours. Inactivate Msp I at 65&deg;C for 10 minutes, then precipitate the DNA in ethanol with 1 &mu;l of 5 mg/ml glycogen. Dissolve the DNA pellet in 50 &mu;l of sterile distilled water.</li>
	<li>Mix 50 &mu;l of Msp I-treated DNA with 2 &mu;l of a 20 &mu;M linker oligonucleotide L (5'-gctgaccctgaattcgcacgtgcctgtcgttagcggacacagggcgattcac-3'), 1 &mu;l of a 20 &mu;M oligonucleotide S (5'-cggtgaatc-3'), 1 &mu;l of sterile distilled water and 6 &mu;l of 10 x T4 DNA ligase buffer. Cover the mixture with liquid wax.</li>
	<li>Denature oligonucleotides at 50&deg;C for 1 minute and allow to cool down gradually to 10&deg;C in a 0.5&deg;C /minute gradient in a thermal cycler.</li>
	<li>Add 1 &mu;l of 400 U/&mu;l T4 DNA ligase and incubate at 15&deg;C overnight. Purify the linker-ligated DNA using a QIAquick PCR Purification kit, and elute in 50 &mu;l of sterile distilled water.</li>
</ol>
7. PCR amplification and sequence analysis
<ol>
	<li>Choose a primer for the specific region of DNA that you wish to examine for long-range interactions (i.e., the 'bait'). In this example, we use ABL-1.</li>
	<li>Take 1 &mu;l of the purified DNA to perform the first round PCR using 1 &mu;l of 20 &mu;M ABL-1 specific primer #4656 (5'-gttcaagcgattctcctgcctcga-3'), 1 &mu;l of 20 &mu;M linker specific primer #2963 (5'-gctgaccctgaattcgcacgtgcct-3'), 3&mu;l of 3 x Klen Taq DNA polymerase I cocktail and 3 &mu;l of sterile distilled water. Two &mu;l of 32P-dCTP is added to 100 &mu;l of 3 x Klen Taq DNA polymerase I cocktail before PCR amplification. The thermal cycle for a hot start PCR is 72&deg;C for 2 min, 95&deg; Cfor 2 minutes; 18 cycles of 95&deg;C for 20 seconds, 65&deg;C for 40 seconds, and 72&deg;C for 1 minute; the final extension is performed at 72&deg;C for 5 minutes.</li>
	<li>Purify the first round PCR products using a QIAquick PCR Purification kit, and elute DNA in 30 &mu;l of sterile distilled water.</li>
	<li>Take 1 &mu;l of 100 x diluted first PCR product to perform a second round of PCR using nested primers by adding 1 &mu;l of 20 &mu;M ABL-1 specific primer #4626 (5'-ggagaatttttatctgcctctgtga-3') (Figure 1a),1 &mu;l of 20 &mu;M of linker specific primer #2961 (5'-gtcgttagcggacacagggcgattc-3'), 3 &mu;l of 3 x Klen Taq DNA polymerase I cocktail and 3 &mu;l of sterile distilled water. The thermal cycling schedule is 25 cycles of 95&deg;C for 20 seconds, 67&deg;C for 40 seconds, and 72&deg;C for 1 minute, followed by extension at 72&deg;C for 5 minutes.</li>
	<li>Visualize the PCR products by running a 5% urea-PAGE gel and scanning the exposed screen in a PhosphoImager (Figure 1b). Each PCR band can be recycled from the gel by dissolving the gel strips in an Eppendorf tube containing 60 &mu;l of sterile distilled water, and incubating at 95&deg;C for 5 minutes. Centrifuge briefly at 10,000 rpm for 10 sec to collect all the samples. Remove 1 &mu;l to use as the DNA template to perform PCR with the primer pair 2961/4626 using the same conditions as described above. Sequence analysis can be performed after purification using a QIAquick PCR Purification kit.</li>
	<li>DNA sequences are analyzed using an online tool to determine their chromosomal location at the web site:http://genome.ucsc.edu(Figure 1c). Click 'BLAT' to enter a new window which allows to paste a DNA sequence, a new window appears after clicking 'submit', and shows the 'BLAT Search Results'. Click 'browser' of the hit with 100% identity to enter a next window to show the location of the DNA sequence.</li>
</ol>
8. Representative Results
1. ACT assay using ABL-1 region as bait to determine its long range DNA interactions
As illustrated in Figure 1a, two Bgl II sites and one BamH I site were chosen for the ACT assay. In the second round of PCR, primer set 4626/2961 was used to amplify ABL-M1, 4630/2961 was used for ABL-M2, and 4636/2961 was used for ABL-M3. A typical gel pattern shows one to several bands (Figure 1b). Each fragment from an ACT assay consists of two combined DNA segments: one segment is derived from the bait DNA region, while the second segment comes from the associated partner, which was joined to the bait region segment by the first restriction enzyme recognition sequence. The second enzyme recognition sequence will appear at the end of the associated partner sequence (Figure 1c). The cloned ABL-M1 fragment contains DNA from the region of ABL1 located at chromosome 9q32.4 from +133,592,306-133,592,399, and the associated partner is located at chromosome 3p13 from +71,869,882-71,870,107. The identities of the associated partners are discovered by blasting their sequences using the UCSC genome browser (GRCh7/hg19 released in February, 2009). PROK2 was identified as the associated partner at the chr3p13 locus.
Similarly, the ABL-M2 associated partner was localized to chr5q21.1, while clone ABL-M3 was identified as an intra-chromosomal association near the ABL-1 locus.
2. Determining less prevalent long range interactions using ACT assay
Other assays based on 3C have reported many more interacting partners than that we have shown in Figure 1 and at the Igf2/H19 locus12. The methodology that we have outlined will select the most prevalent long range interactions. However, by increasing the number of PCR cycles, it is possible to identify additional, less frequent associations as well (Figure 2).
3. Differences in long range interactions in cancer cells when compared to normal tissues.
The ACT assay can also be used to identify differences in nuclear architecture and long range interactions between normal cells and cancer cells (Figure 3). These differences may reflect nuclear architecture changes that occur during cell transformation, and thus this assay may ultimately be applicable for diagnostic purposes. The similar gel patterns that occur in both normal and cancer tissues indicated that the ACT assay is reliable and repeatable. While the ACT assay can identify long range DNA partners, further analyses, such as FISH and ChIP assays, are required to verify the presence of the identified associations between distant loci. Genetic, physiological and biochemical studies can then be performed to elucidate the biological implications of these long range DNA associations.
<img src="/files/ftp_upload/2621/2621fig1.jpg" alt="Figure 1" /> 
Figure 1 ACT assay using ABL-1 region as bait DNA in HL-60 cells. a. DNA structure in ABL-1 region. The first restriction enzyme used in ACT assay was Bgl II. The primers for the second round of PCR are also labeled by arrows and corresponding primer numbers. b. Gel pattern of the ACT assay in 5% urea-PAGE. Primer pair 4626/2961 was used for amplifying clone ABL-M1 in the nested PCR, 4630/2961 was used for clone ABL-M2, and 4636/2961 was used for clone ABL-M3. c. DNA sequence of clone ABL-M1. DNA fragment from ABL-1 bait DNA is labeled in red, and the associated DNA partner is labeled in cyan. Bgl II site (AGACTC) was labeled in green, and Msp I site (CCGG) is labeled in purple.
<img src="/files/ftp_upload/2621/2621fig2.jpg" alt="Figure 2" /> 
Figure 2 PCR cycles affect ACT assay results. Using the imprinting control region (ICR) at the Igf2/H19 locus as bait DNA in mouse fibroblast cells, different cycling programs were applied in the first and second rounds of PCR in the ACT assay. 18-20 cycles in the first round of PCR did not amplify enough signal to be visualized. Twenty five cycles in the second PCR results in clear bands, while increasing the number of cycles for the second round of PCR induced a smear pattern in addition to more bands.
<img src="/files/ftp_upload/2621/2621fig3.jpg" alt="Figure 3" /> 
Figure 3 Detection of differences in nuclear architecture between normal colon tissue and colon cancer tissue in ACT assay. a. DNA structure of the ICR region at IGF2/H19 locus and IGF2 gene. Dpn II sites for ACT assay are labeled. Primers used in the second round PCR are labeled by arrows and numbers in different colors which correspond to each lane in panel b of the figure. b. Gel pattern of the ACT assay in 5% urea-PAGE. Normal colon tissue MAD03-1423 and colon cancer tissue MAD04-149 was obtained from Cooperative Human Tissue Network (CHTN) Western Division. After each colon tissue was homogenized, ACT assay was performed following the procedures described herein. Lane 1 represents the PCR results using primer pair #2961and #4161(5'-tctgcgccatcagggcagtgagac-3') labeled in pink in panel a; lane 2 represents the PCR results using primer pair #2961 and #4163 (5'-gccgcgcggccacttccgattcc-3') labeled in orange in panel a; lane 3 represents the PCR results using primer pair #2961 and #5145 (5'-gccatgcaggtaggatttgagctg-3') labeled in blue in panel a; lane 4 represents the PCR results using primer pair #2961 and #5151(5'-gtctcaaataggggccagctagcttgg-3') labeled in green in panel a. Unique bands that appear only in normal colon tissue are labeled by yellow arrows, and those bands that appeared only in colon cancer tissue are labeled in red arrows. ICR, imprinting control region; DMR, different methylated region.

<ol>
	<li>Dekker, J., Rippe, K., Dekker, M., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+chromosome+conformation.">Capturing chromosome conformation.</a> Science. 295, 1306-1311 (2002).</li><li>Apostolou, E., Thanos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+Infection+Induces+NF-kappaB-dependent+interchromosomal+associations+mediating+monoallelic+IFN-beta+gene+expression.">Virus Infection Induces NF-kappaB-dependent interchromosomal associations mediating monoallelic IFN-beta gene expression.</a> Cell. 134, 85-96 (2008).</li><li>Duan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+model+of+the+yeast+genome.">A three-dimensional model of the yeast genome.</a> Nature. 465, 363-367 (2010).</li><li>Lomvardas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchromosomal+interactions+and+olfactory+receptor+choice.">Interchromosomal interactions and olfactory receptor choice.</a> Cell. 126, 403-413 (2006).</li><li>Murrell, A., Heeson, S., Reik, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+differentially+methylated+regions+partitions+the+imprinted+genes+Igf2+and+H19+into+parent-specific+chromatin+loops.">Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.</a> Nat Genet. 36, 889-893 (2004).</li><li>Schoenfelder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+associations+between+co-regulated+genes+reveal+a+transcriptional+interactome+in+erythroid+cells.">Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.</a> Nat Genet. 42, 53-61 (2010).</li><li>Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchromosomal+associations+between+alternatively+expressed+loci.">Interchromosomal associations between alternatively expressed loci.</a> Nature. 435, 637-645 (2005).</li><li>Vakoc, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+among+distant+regulatory+elements+at+the+beta-globin+locus+requires+GATA-1+and+FOG-1.">Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.</a> Mol Cell. 17, 453-462 (2005).</li><li>Xu, N., Tsai, C. L., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+homologous+chromosome+pairing+marks+the+onset+of+X+inactivation.">Transient homologous chromosome pairing marks the onset of X inactivation.</a> Science. 311, 1149-1152 (2006).</li><li>Lieberman-Aiden, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 326, 289-293 (2009).</li><li>Berkum, N. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hi-C:+a+method+to+study+the+three-dimensional+architecture+of+genomes.">Hi-C: a method to study the three-dimensional architecture of genomes.</a> J Vis Exp. , (2010).</li><li>Ling, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTCF+mediates+interchromosomal+colocalization+between+Igf2/H19+and+Wsb1/Nf1.">CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1.</a> Science. 312, 269-272 (2006).</li><li>Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three+'C's+of+chromosome+conformation+capture:+controls,+controls,+controls.">The three 'C's of chromosome conformation capture: controls, controls, controls.</a> Nat Methods. 3, 17-21 (2006).</li></ol>

No conflicts of interest declared.

Dekker et al. developed the chromosome conformation capture (3C) approach to detect the frequency of interaction between two genomic loci1, and 3C has been used extensively to investigate intra-chromosomal and inter-chromosomal associations between two known DNA regions in mammalian cells2-9. Although newly developed Hi-C methodology can be applied for study of genome-wide DNA association, ACT assay is still an effective technique for study of locus-specific DNA interaction10-11. We have modified this approach to identify unknown DNA regions that are associated with a known DNA region in cultured mouse and human cells (Figure 1). We named this method the associated chromosome trap (ACT) assay, as it provided us a reliable and reproducible method to identify novel unknown DNA partners that associate with a known target DNA region12. A successful 3C assay with appropriate controls is performed before executing the novel aspects of the ACT assay13. In order tofind as many associated DNA regions as possible, it is necessary to use various combinations of first and second restriction enzymes. It is especially important to use restriction enzymes that are insensitive to CpG methylation to perform the first 3C ligation step. Protein binding and DNA methylation can also influence restriction enzyme digestion efficiency and can lead to failure of ligation of the associated DNA regions for certain restriction enzyme digestions. The occurrence of intra- or inter-chromosomal ligation depends on protein-DNA cross-linking and appropriate physical maps of both of the associated DNA regions. Thus, some preliminary experiments are essential to establish a practicable effective formaldehyde concentration and treatment time in the ACT assay. A range of final concentrations of formaldehyde (from1.5% to 2%) have been used in several labs during the 3C portion of the assay7,9. Alternatively, the oligonucleotides used as linkers can be designed to match the cohesive end cut by the second restriction enzyme. Although we found that 18-20 cycles in the first round of PCR and 20-25 cycles in the second round of PCR could provide clear bands, it is necessary to establish the best PCR conditions for each experiment (Figure 2).Intra-molecule annealing between 5'-end and 3'-end complementary adaptor sequence of a DNA strand may occur in PCR, it inhibits adaptor-specific primer annealing with the DNA molecule, and resulted in much lower amplification efficiency in the first several cycles. After the target DNA was amplified for cycles, its amount may be much larger than these nonspecific reactions, and may facilitate the competition of primer annealing to the target DNA molecules. This is also why we may see background amplification, and why the first round PCR product has to be diluted to decrease background amplification.It is important to remove excess primers from the PCR reaction in order to decrease the background in the second round of PCR. As in all PCR-based experiments, it is vital to design primers that are not located in repeat sequence regions, which constitute the majority of human and mouse DNA. Although newly developed Hi-C methodology can be applied for study of genome-wide DNA association, ACT assay is still an effective technique for study of locus-specific DNA interaction.

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<td>acrylamide</td>
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<td>&nbsp;</td>
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<td>AM8110G</td>
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<td>fetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
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<td>&nbsp;</td>
<td>Invitrogen</td>
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<td>Invitrogen</td>
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<td>NEB Biolabs</td>
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<td>NEB Biolabs</td>
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associated chromosome trap for identifying long-range dna interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

Watch this Scientific Journal Video about Associated Chromosome Trap for Identifying Long-range DNA Interactions at JoVE.com

Associated Chromosome Trap for Identifying Long-range DNA Interactions

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

associated-chromosome-trap-for-identifying-long-range-dna-interactions

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Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...