This work was supported in part by the Rhode Island Institutional Development Award (IDeA) Network of Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number&#160;P20GM103430 and the Bryant Center of Health and Behavioral Sciences.

Chromosome Conformation Capture (3C) for Undergraduate Research and Teaching

<ol>
	<li>McBryant, S. J., Adams, V. H., Hansen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+architectural+proteins.">Chromatin architectural proteins.</a> Chromosome Research. 14 (1), 39-51 (2006).</li><li>Nalabothula, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromatin+architectural+proteins+HMGD1+and+H1+bind+reciprocally+and+have+opposite+effects+on+chromatin+structure+and+gene+regulation.">The chromatin architectural proteins HMGD1 and H1 bind reciprocally and have opposite effects on chromatin structure and gene regulation.</a> BMC Genomics. 15, 92 (2014).</li><li>John, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+accessibility+pre-determines+glucocorticoid+receptor+binding+patterns.">Chromatin accessibility pre-determines glucocorticoid receptor binding patterns.</a> Nature Genetics. 43 (3), 264-268 (2011).</li><li>Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+occurrence+of+a+fibrous+lamina+on+the+inner+aspect+of+the+nuclear+envelope+in+certain+cells+of+vertebrates.">On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates.</a> The American Journal of Anatomy. 119 (1), 129-145 (1966).</li><li>Reddy, K. L., Zullo, J. M., Bertolino, E., Singh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+repression+mediated+by+repositioning+of+genes+to+the+nuclear+lamina.">Transcriptional repression mediated by repositioning of genes to the nuclear lamina.</a> Nature. 452 (7184), 243-247 (2008).</li><li>Finlan, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+to+the+nuclear+periphery+can+alter+expression+of+genes+in+human+cells.">Recruitment to the nuclear periphery can alter expression of genes in human cells.</a> PLoS Genetics. 4 (3), e1000039 (2008).</li><li>Chambeyron, S., Da Silva, N. R., Lawson, K. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+re-organisation+of+the+Hoxb+complex+during+mouse+embryonic+development.">Nuclear re-organisation of the Hoxb complex during mouse embryonic development.</a> Development. 132 (9), 2215-2223 (2005).</li><li>Chambeyron, S., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+decondensation+and+nuclear+reorganization+of+the+HoxB+locus+upon+induction+of+transcription.">Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.</a> Genes and Development. 18 (10), 1119-1130 (2004).</li><li>Mahy, N. L., Perry, P. E., Gilchrist, S., Baldock, R. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+of+active+and+inactive+genes+and+noncoding+DNA+within+chromosome+territories.">Spatial organization of active and inactive genes and noncoding DNA within chromosome territories.</a> The Journal of Cell Biology. 157 (4), 579-589 (2002).</li><li>Mahy, N. L., Perry, P. E., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+density+and+transcription+influence+the+localization+of+chromatin+outside+of+chromosome+territories+detectable+by+FISH.">Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH.</a> The Journal of Cell Biology. 159 (5), 753-763 (2002).</li><li>Li, B., Carey, M., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chromatin+during+transcription.">The role of chromatin during transcription.</a> Cell. 128 (4), 707-719 (2007).</li><li>Chen, A., Chen, D., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+of+DNase-seq+for+mapping+active+gene+regulatory+elements+across+the+genome+in+animals.">Advances of DNase-seq for mapping active gene regulatory elements across the genome in animals.</a> Gene. 667, 83-94 (2018).</li><li>Hughes, A. L., Rando, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+nucleosome+positioning+in+vivo.">Mechanisms underlying nucleosome positioning in vivo.</a> Annual Review of Biophysics. 43, 41-63 (2014).</li><li>Weiner, A., Hughes, A., Yassour, M., Rando, O. J., Friedman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+nucleosome+mapping+reveals+transcription-dependent+promoter+packaging.">High-resolution nucleosome mapping reveals transcription-dependent promoter packaging.</a> Genome Research. 20 (1), 90-100 (2010).</li><li>Hughes, A. L., Jin, Y., Rando, O. J., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+evolutionary+approach+to+identify+determinants+of+nucleosome+positioning:+A+unifying+model+for+establishing+the+genome-wide+pattern.">A functional evolutionary approach to identify determinants of nucleosome positioning: A unifying model for establishing the genome-wide pattern.</a> Molecular Cell. 48 (1), 5-15 (2012).</li><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 (5558), 1306-1311 (2002).</li><li>van Berkum, N. L., 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=Determining+spatial+chromatin+organization+of+large+genomic+regions+using+5C+technology.">Determining spatial chromatin organization of large genomic regions using 5C technology.</a> Methods in Molecular Biology. 567, 189-213 (2009).</li><li>Smith, E. M., Lajoie, B. R., Jain, G., 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=Invariant+TAD+boundaries+constrain+cell-type-specific+looping+interactions+between+promoters+and+distal+elements+around+the+CFTR+locus.">Invariant TAD boundaries constrain cell-type-specific looping interactions between promoters and distal elements around the CFTR locus.</a> American Journal of Human Genetics. 98 (1), 185-201 (2016).</li><li>Crane, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+inputs+into+C.+elegans+dosage+compensation:+Chromosome+conformation+and+the+miRNA-specific+argonaute+ALG-2.">Two inputs into C. elegans dosage compensation: Chromosome conformation and the miRNA-specific argonaute ALG-2.</a> University of California, Berkeley. , (2012).</li><li>Bell, A. C., West, A. G., Felsenfeld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+CTCF+is+required+for+the+enhancer+blocking+activity+of+vertebrate+insulators.">The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.</a> Cell. 98 (3), 387-396 (1999).</li><li>Cuadrado, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+contributions+of+cohesin-SA1+and+cohesin-SA2+to+TADs+and+polycomb+domains+in+embryonic+stem+cells.">Specific contributions of cohesin-SA1 and cohesin-SA2 to TADs and polycomb domains in embryonic stem cells.</a> Cell Reports. 27 (12), 3500-3510 (2019).</li><li>Sarro, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+the+three-dimensional+regulatory+topology+of+the+Pitx1+locus+results+in+overtly+normal+development.">Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development.</a> Development. 145 (7), (2018).</li><li>Tolhuis, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+among+polycomb+domains+are+guided+by+chromosome+architecture.">Interactions among polycomb domains are guided by chromosome architecture.</a> PLoS Genetics. 7 (3), e1001343 (2011).</li><li>Splinter, E., de Wit, E., van de Werken, H. J. G., Klous, P., de Laat, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+long-range+chromatin+interactions+for+selected+genomic+sites+using+4C-seq+technology:+From+fixation+to+computation.">Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: From fixation to computation.</a> Methods. 58 (3), 221-230 (2012).</li><li>Fern&#225;ndez-Mi&#241;&#225;n, A., Bessa, J., Tena, J. J., G&#243;mez-Skarmeta, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+21+-+Assay+for+transposase-accessible+chromatin+and+circularized+chromosome+conformation+capture,+two+methods+to+explore+the+regulatory+landscapes+of+genes+in+zebrafish.">Chapter 21 - Assay for transposase-accessible chromatin and circularized chromosome conformation capture, two methods to explore the regulatory landscapes of genes in zebrafish.</a> Methods in Cell Biology. 135, 413-430 (2016).</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+&amp;#34;C&amp;#34;+s+of+chromosome+conformation+capture:+controls,+controls,+controls.">The three &amp;#34;C&amp;#34; s of chromosome conformation capture: controls, controls, controls.</a> Nature Methods. 3 (1), 17-21 (2006).</li><li>Hag&#232;ge, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+chromosome+conformation+capture+assays+(3C-qPCR).">Quantitative analysis of chromosome conformation capture assays (3C-qPCR).</a> Nature Protocols. 2 (7), 1722-1733 (2007).</li><li>Lajoie, B. R., van Berkum, N. L., Sanyal, A., 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=My5C:+Webtools+for+chromosome+conformation+capture+studies.">My5C: Webtools for chromosome conformation capture studies.</a> Nature Methods. 6 (10), 690-691 (2009).</li><li>Han, M., Wei, G., McManus, C. E., Hillier, L. W., Reinke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+C.+elegans+germ+nuclei+exhibit+distinct+genomic+profiles+of+histone+modification+and+gene+expression.">Isolated C. elegans germ nuclei exhibit distinct genomic profiles of histone modification and gene expression.</a> BMC Genomics. 20 (1), 500 (2019).</li></ol>

The authors declare no conflicts of interest.

The 3C is a powerful technique that is rooted in basic molecular techniques. It is this foundation of fundamental tools that makes 3C such an intriguing technique to use with undergraduates. With so many recent studies observing chromatin dynamics on such a broad scale, using these results to devise a narrow-focused experiment on a single gene or genomic region has the potential to create a unique and impactful experiment in undergraduate research. Often, experiments like these are considered too advanced for undergraduates, but with careful planning, they are easily achievable. It is important to note that the assays designed to probe the chromatin connections captured by the 3C library can vary from semi-quantitative endpoint PCR to whole-genome sequencing. In fact, data from the first 3C paper16 were generated from qPCR. This wide range of assays can all be used because all 3C technologies produce the same product-a library of DNA fragments representing 3D connections in the nucleus.
Presented here is an adaptation of a more flexible and accommodating protocol that is a better fit for undergraduate researchers. The pausing periods listed above imply overnight delays; however, these pauses can extend over weekends and, in the case of the cells and nuclei, for weeks. The most crucial consideration is when the work will get done. Often in protocols, there are time-sensitive steps when pausing is not an option. Outside of a few points (day 1 and day 2), there are many places to stop and freeze the sample. These are critical when working with undergraduates where the schedules and timings of lab work need to be flexible. In addition to engineering these stops into the protocol, undergraduates are encouraged to work in pairs or even small groups of three or four. Groups work well for this protocol, as the students can support each other and create a buddy system so everyone is working safely. Lab work is also more fun with others involved. With groups, students can also work on a variety of questions focused on the organization of chromatin while still performing the same protocol. Thus, even while students are working on separate projects, the protocol links their efforts, and because of this, they can support each other.
Other adaptations are meant to work around the fact that certain specialized tools and equipment are not necessarily found in all undergraduate institutions. These pieces of equipment include but are not limited to qPCR thermocyclers, gel documentation systems, and nano volume spectrometers. Indeed, these pieces of equipment are convenient but are not a requirement. Here, the classic method of 3C is also described in the primer design portion; this involves identifying a genomic locus of interest and, from that, assessing other genomic loci for chromatin contact points further away. This technique also works well if a published dataset is used, such as a dataset using Hi-C, where known positive (connecting) and negative (non-connecting) loci are identified. Designing experiments using these published data sets is another great adaptation for teaching labs, as the chance for the successful identification of chromatin connections is usually greater. In addition, the research article can be discussed in class and be used as a reference.
This protocol uses a modified qPCR approach to visualize the 3C product formation. Controls are essential to the success of the 3C technique. Each experiment uses both sample controls and primer controls to determine the completion of the 3C procedure. The sample controls include an undigested control (genomic DNA) and digested control. The undigested control determines the baseline signal for the primer sets and is used with the cross-linked digestion control to determine the digestion efficiency It is expected that there will be a drop in product for any primers directed across a restriction site. Comparing this value to the undigested control provides an indication of how well the sample was digested.
The primers for the PCR include a control primer and test primers. The control primer is a primer set that is near the genomic region being assayed and does not contain a restriction site. This provides the baseline for determining the abundance of the test primer PCR products. Test primers are forward and reverse primers that flank a restriction site for a particular genomic locus of interest (Figure 3). Reactions using these primer sets are compared to determine the digestion efficiency, as the product abundance should drop if the restriction site has been cut. In determining the chromatin organization, one test primer from one locus is paired with another test primer from a different genomic locus to determine if these two loci are close together in 3D space. In that case, the expectation is that a PCR product would only be found using the 3C sample as the template.
It is important to note that even validated primers have the tendency to fail (Figure 4: r14 primer set). In addition, PCR products are frequently identified in control reactions and in reactions in which a chromatin connection is not predicted (such as the digested control, since it is not ligated). These instances are sequenced and either fail Sanger QC or return without a defined sequence (Figure 5). Additionally, traditional 3C experiments generate a &#34;control template,&#34; an uncross-linked, digested, and ligated DNA sample, which represents all the possible ligated fragments that can be produced with a given amount of DNA. The &#34;control template&#34; plays an important role in comparing the intensities of qPCR signals between two genomic loci to determine if the signal represents a true interaction or just a random association. Creating a &#34;control template&#34; can be problematic, as a large portion of the chromatin being assayed must be captured in the form of an artificial chromosome and processed along with the 3C samples. Securing such a construct may not be feasible, and creating one could be outside the scope of a semester project. Due to these difficulties, we suggest using a control primer. The control primer does not replace all of the functionality of the &#34;control template&#34; but still provides the opportunity to analyze the data to make &#34;presence&#34; or &#34;absence&#34; determinations.
When performing qPCR, using equal amounts of the sample is important. This should be determined, even if using a nano-spectrophotometer such as a nanodrop, by generating a standard curve from genomic DNA of a known concentration and fitting the 3C samples to that line. These amounts should be recorded and used in subsequent PCRs. The quality of the PCR reaction is also important. As the PCR runs in qPCR, the product abundance is measured using fluorescence and recorded. This recording is accessible in the amplification plot. Once the program has finished, it is important to check the amplification plot and ensure that the reactions (except for the no template controls) have three phases: a baseline, an exponential, and a plateau/saturation phase. It is important to check that the reactions have an exponential phase, in particular for setting the threshold (see below). Additionally, for serially diluted samples, there should be a shift in Ct values consistent with the dilution of the sample (the highest concentration will have the lowest Ct values, while the lowest concentration will have the highest Ct values). Samples that do not reflect this change in the amplification plot require a new dilution or indicate a larger issue with the 3C sample formation. Finally, while generating the standard curve, the PCR software will compute the PCR efficiency and the R2 value. The PCR efficiency should be greater than 90%, and the R2 value should be greater than 0.99. If either of these conditions are not met, it is likely that something is wrong with the sample or the PCR primers.
Post qPCR, the percent digestion and the presence of 3C interactions can be calculated using the qPCR Ct for each reaction. To determine these, first, the threshold for the PCR reaction must be set. This is normally done using the software that comes with the qPCR machine. Setting the threshold will define the concentration of PCR product that will be used to compare the sample Ct values. The threshold should bisect the amplification curves of the PCR reactions in the exponential phase of amplification. Only PCR reactions with exponential amplification can be compared (in this case, the control primers and the test primer reactions), as this is the only way to ensure the reactions are amplifying DNA at the same rate and can be compared faithfully. When analyzing the 3C graphs, conditionally positive reactions are identified as those with more product over the control samples, genomic control, and the digestion control (Figure 4B). However, these samples must be further validated using Sanger sequencing following the gel purification of the PCR product.
After Sanger sequencing, samples that pass the QC can be analyzed using Blat. The goal of this analysis is to determine if the sample has the sequence of both target genomic loci flanking the restriction site (DpnII in the case of this protocol). If both sequences are identified, then the 3C fragment can be considered validated. If the results of the Blat do not return the expected sequence, this may indicate that one or both primers are not optimal, resulting in a false positive qPCR result. The trace files for the false positive samples will have undefined base peaks, and the Seq reports will contain mostly &#34;n&#34; base calls.
Sanger validation is essential, as false positives from artifactual PCR product formation are possible. These false positives can be identified when the sequencing products do not have the expected target sequence or a DpnII site characteristic of a proper 3C fragment (Figure 5). The sequencing of the PCR fragments also provides another data point for the experiment and drives home to the students that the 3C technique is identifying distant genomic loci that are coming together in 3D space within the nuclei.
The 3C technique provides a wealth of foundational molecular techniques for undergraduates in a flexible, straightforward procedure. This 3C technique is also a launching point for the other 3C techniques that incorporate next-generation sequencing (NGS). These types of experiments can expose undergraduates to important aspects of bioinformatics and are rooted in the basic principles outlined here. Undergraduate experience and involvement are key to their success and development as young scientists. By providing these opportunities, undergraduates can strengthen their understanding of basic principles while building their confidence to tackle cutting-edge techniques and questions.

An organism&#39;s genome not only holds all the genes required for function but also all the instructions on how and when to use them. This makes regulating access to the genome one of the most important functions of the cell. There are many mechanisms to control gene function; however, at its base level, gene regulation comes down to the ability of regulatory transcription factors (trans-factors) to bind to their specific DNA sequences (cis-regulatory sequences). This is not an innate ability; instead, it is governed by the organization/structure of the genome in the nucleus, which controls the availability/exposure of the cis-regulatory sequences to the trans-factors1,2,3. If the trans-factors cannot find their cis-regulatory sequences, then the trans-factors cannot perform their regulatory tasks. This has made understanding how genomes are organized in the nucleus an important source of inquiry.
It is widely accepted that during interphase, eukaryotic chromosomes in the nucleus occupy their own domain anchored to the nuclear lamina and nuclear matrix (Figure 1), thus making the chromosome more like a slice of pizza, rather than a noodle on a plate of spaghetti. Chromosomes are partially condensed by protein-DNA interactions (chromatin) that twist and loop portions of the chromosome. Through electron microscopy, three-dimensional DNA fluorescence in situ hybridization (FISH), and DNA tagging techniques (i.e., fluorescent and artificial DNA methylation), inactive domains of chromatin have been found to be packed tightly along the nuclear periphery4,5,6, while portions of active, less condensed chromatin are found in the interior of the nucleus7,8,9,10. These experiments provide a wide-angle view of chromosome dynamics but do little to capture the changes that occur locally around the gene promotors observed in DNase11,12 and nucleosome13,14,15 studies.
The key to unlocking higher-resolution chromatin dynamics was the formulation of the 3D chromosome mapping technique, 3C. The 3C technique itself comprises four main steps: crosslinking of chromatin, chromatin digestion by restriction enzymes, chromatin ligation, and DNA purification (Figure 2). The new artificial DNA fragments generated by this process can then be characterized to reveal the close physical association between linearly distant pieces of DNA16. The 3C technique became the basis for the creation of multiple spin-off techniques that utilize the initial steps of 3C to ask broader genome-wide questions (e.g., Hi-C, 4C, ChIP-C). This family of 3C techniques has identified that chromosomes are organized into multiple discrete units termed topologically associated domains (TADs). TADs are encoded in the genome and are defined by chromatin loops flanked by unlooped boundaries16,17,18,19. The TAD boundaries are maintained by two evolutionarily conserved and ubiquitous factors, including CCCT binding factor (CTCF) and cohesion, which prevent loops within separate TADs from interacting16,20. The loops are mediated by the interaction of trans-factors with their regulatory sequences, as well as CTCF and cohesion21.
Though many studies using 3C technologies ask broad genome-wide questions and employ complicated sample collection techniques, the formulation of the 3C technique is based on basic molecular biology techniques. This makes 3C intriguing for deployment in both undergraduate research and teaching labs. The 3C technique can be employed for smaller focused questions and is inherently flexible to scaling up or down (single genes22, chromosomes16, and/or genomes18) depending on the focus and direction of the questions asked. This technique has also been applied to a wide range of model systems7,16,19,23 and has been proven to be versatile in its use. This makes 3C an excellent technique for undergraduates in that students can gain experience in common molecular biology techniques while also gaining valuable experience in answering directed questions.
Presented here is an adapted protocol for 3C library preparation based on previously published protocols24,25,26,27. This protocol has been optimized for approximately 1 &#215; 107 cells, though it has generated 3C libraries with as little as 1 &#215; 105 cells. This protocol has proven to be versatile and has been used to generate 3C libraries from zebrafish embryos, zebrafish cell lines, and young-adult (YA) Caenorhabditis elegans (roundworm). The protocol should also be appropriate for mammalian cell lines and, with further adaptation, yeast.
The goal of these adaptations is to make 3C more accessible for undergraduates. Care has been taken to use techniques that are similar to those that can be accomplished in an undergraduate teaching laboratory. The 3C technique provides many learning opportunities for undergraduates to learn basic molecular biology techniques that will benefit their development at the bench, in the classroom, and in their endeavors after graduation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37% Formaldehyde&#160;</td>
			<td>Millapore-Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Millapore-Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>MP Biomedical</td>
			<td>215350280</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Millapore-Sigma</td>
			<td>C0549</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Millapore-Sigma</td>
			<td>COEDTAF-RO</td>
			<td>mixed to 50x in water. Diluted to 1x in Sucrose buffer and GB buffer fresh</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Millapore-Sigma</td>
			<td>D0632</td>
			<td>1 M stock diluted to 500 &#181;M in Sucrose buffer and GB buffer fresh</td>
		</tr>
		<tr>
			<td>DpnII</td>
			<td>NEB</td>
			<td>R0543M</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Millapore-Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Millapore-Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>glycogen</td>
			<td>Millapore-Sigma</td>
			<td>10901393001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millapore-Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millapore-Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Millapore-Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl green pyronin&#160;&#160;</td>
			<td>Millapore-Sigma</td>
			<td>HT70116</td>
			<td></td>
		</tr>
		<tr>
			<td>MgAc2</td>
			<td>Thermoscientific</td>
			<td>1222530</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Millapore-Sigma</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Millapore-Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol-chloroform&#160;</td>
			<td>Millapore-Sigma</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Millapore-Sigma</td>
			<td>11459643001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>IBI Scientific</td>
			<td>IB05406</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Ready mix (Phire Taq etc)</td>
			<td>Millapore-Sigma</td>
			<td>KCQS07</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A&#160;</td>
			<td>Millapore-Sigma</td>
			<td>R6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Millapore-Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate (SDS)</td>
			<td>Millapore-Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millapore-Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Millapore-Sigma</td>
			<td>108319</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millapore-Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</td>
			<td>Millapore-Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

getting an a with the 3cs: chromosome conformation capture for undergraduates

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C techniques) that provide detailed information of the three-dimensional organization of chromatin. The 3C techniques have been used in a wide range of studies, from monitoring the changes in chromatin organization in cancer cells to identifying enhancer contacts made with gene promoters. While many of the studies using these techniques are asking big genome-wide questions with intricate sample types (i.e., single-cell analysis), what is often lost is that the 3C techniques are grounded in basic molecular biology methods that are applicable to a broad range of studies. By addressing tightly focused questions of chromatin organization, this cutting-edge technique can be used to enhance the undergraduate research and teaching lab experience. This paper presents a 3C protocol and provides adaptations and points of emphasis for implementation at primarily undergraduate institutions in undergraduate research and teaching experiences.

This procedure will produce one experimental 3C sample and two control samples (undigested and digested). Using these three samples, qPCR was performed. From these results, the digestion efficiency was calculated (equation 1) and recorded (Table 1). From these calculations, it was determined that the 3C sample had an approximately 88% digestion efficiency (average of Table 1) across the seven genomic loci tested.
Next, the samples were tested for the presence of long-range chromatin contacts between the different genomic loci using combinations of loci-specific primers (Table 2) and qPCR. Using these results, the product abundances relative to a control primer set were calculated (equation 2) and graphed for comparison (Figure 4). These data indicated that 8 of the 10 reactions were conditional positives for long-range interactions.
The PCR reactions were then run on an agarose gel. The expected PCR product for the eight conditional positives and one negative reaction were gel-purified and sent for Sanger sequencing. The results for representative positive (blue arrow, blue box) and negative (red arrow, red box) reactions are shown (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65213/65213fig01.jpg" /> 
Figure 1: Chromosome structure in the nucleus. Hypothetical chromosome organization inside the nucleus. (A) Nuclear envelope, black lines; (B) nuclear lamina, orange; (C) heterochromatin, compacted lines; (D) euchromatin, loose loops. <a href="https://www.jove.com/files/ftp_upload/65213/65213fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65213/65213fig02.jpg" /> 
Figure 2: Schematic of the 3C protocol. Distant portions of linear chromosomes (blue, yellow, and pink) are brought close together within the nucleus through regulatory loops. (A) The loop structures are mediated by transcription factors (grey circle and black star); these interactions are preserved through chemical crosslinking. (B) The loops are broken through enzymatic digestion (black lines). (C) Distant chromatin pieces are ligated together through the sticking ends created by the digestion. (D) DNA is purified from protein. (E) The sequence within the fragments is identified and mapped back to the genome. <a href="https://www.jove.com/files/ftp_upload/65213/65213fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65213/65213fig03.jpg" /> 
Figure 3: The 3C primer scheme. The 3C primers are designed around restriction sites at varying distances from the genomic location of interest. The pink arrows represent the experimental primer sets surrounding a DpnII site. The experimental primers can be mixed and matched to assess chromatin looping in the region. The orange primers represent a negative control without a DpnII site. <a href="https://www.jove.com/files/ftp_upload/65213/65213fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65213/65213fig04.jpg" /> 
Figure 4: Representative qPCR data for a 3C experiment. Relative abundance of the 3C test primer sets. (A) Control graph showing the relative abundance of the product in the undigested control (blue), digested control (grey), and 3C sample (orange). (B) The 3C experiment; the relative abundance of the product from combinations of test primers in the undigested control (blue), digested control (grey), and 3C sample (orange). <a href="https://www.jove.com/files/ftp_upload/65213/65213fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65213/65213fig05.jpg" /> 
Figure 5: Visualization of the 3C qPCR products. Top, gel with the qPCR endpoint products with the samples indicated. Bottom, representative Sanger sequence trace files for the reactions indicated. The orange sample is an example of a false positive (see Figure 4, r38/34), and the blue sample is an example of a true positive with the DpnII site indicated above the trace. <a href="https://www.jove.com/files/ftp_upload/65213/65213fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Site</td>
			<td>% digestion</td>
		</tr>
		<tr>
			<td>r8</td>
			<td>86.98</td>
		</tr>
		<tr>
			<td>r3</td>
			<td>88.44</td>
		</tr>
		<tr>
			<td>r38</td>
			<td>89.64</td>
		</tr>
		<tr>
			<td>r34</td>
			<td>87.55</td>
		</tr>
		<tr>
			<td>r33</td>
			<td>87.85</td>
		</tr>
		<tr>
			<td>r14</td>
			<td>86.97</td>
		</tr>
		<tr>
			<td>r47</td>
			<td>89.45</td>
		</tr>
	</tbody>
</table>
Table 1: Calculated digestion efficiency.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td colspan="3">Sequence</td>
			<td>Genomic loci</td>
		</tr>
		<tr>
			<td>r3 FWD</td>
			<td colspan="3">ACGCAAGTAAAATTCTGGTTTTTGACC</td>
			<td>chrX:11361475</td>
		</tr>
		<tr>
			<td>r3 RVS</td>
			<td colspan="3">TTTCCTGAGCTCTAACCATGTTTGC</td>
			<td>chrX:11361561</td>
		</tr>
		<tr>
			<td>r38 FWD</td>
			<td colspan="3">TTACTTCTGAAGTAATCTTTTCTTATCCCC</td>
			<td>chrX:5859700</td>
		</tr>
		<tr>
			<td>r38 RVS</td>
			<td colspan="3">AGACGAGCTGATTAAAAGTAGTTGAGAG</td>
			<td>chrX:5859775</td>
		</tr>
		<tr>
			<td>r34 FWD</td>
			<td colspan="3">ATTTGTGGATTGCGTGGAGACG</td>
			<td>chrX:5429702</td>
		</tr>
		<tr>
			<td>r34 RVS</td>
			<td colspan="3">AATAATCCTCTTAACAAACGTGGCC</td>
			<td>chrX:5429777</td>
		</tr>
		<tr>
			<td>r33 FWD</td>
			<td colspan="3">AAGAGTTGTCCAAAATAAATTGAGCTAAC</td>
			<td>chrX:6296704</td>
		</tr>
		<tr>
			<td>r33 RVS</td>
			<td colspan="3">TTCAGAAAAGTAAACTTTGACTTGGAACG</td>
			<td>chrX:6296807</td>
		</tr>
		<tr>
			<td>r14 FWD</td>
			<td colspan="3">AATTATCGATTTTTCCATCGCGCAG</td>
			<td>chrX:8036367</td>
		</tr>
		<tr>
			<td>r14 RVS</td>
			<td colspan="3">ATTTCAATGAAAATGTAAAAATGTTCCTTC</td>
			<td>chrX:8036427</td>
		</tr>
		<tr>
			<td>r47 FWD</td>
			<td colspan="3">ATCTAGACTTGATAATATTTGTGTGTCCTC</td>
			<td>chrX:9464939</td>
		</tr>
		<tr>
			<td>r47 RVS</td>
			<td colspan="3">AAGTTCTGCAACTGTTAGATGAATAACAC</td>
			<td>chrX:9465064</td>
		</tr>
		<tr>
			<td>r8 FWD</td>
			<td colspan="3">GAGAATGTTGTTCTGTAACTGAAAACTTG</td>
			<td>chrX:11094257</td>
		</tr>
		<tr>
			<td>r8 RVS</td>
			<td colspan="3">TTACGAAATTTGGTAGTTTTGGACC</td>
			<td>chrX:11094362</td>
		</tr>
		<tr>
			<td>Control primer FWD</td>
			<td colspan="3">CAATCGTCTCGCTCACTTGTC</td>
			<td>chrX:7608049</td>
		</tr>
		<tr>
			<td>Control primer RVS</td>
			<td colspan="3">GATGTGAGCAACAAGGCACC</td>
			<td>chrX:7608166</td>
		</tr>
	</tbody>
</table>
Table 2: Primers for the representative 3C experiment.

Watch this Scientific Journal Video about Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates at JoVE.com

Author Spotlight: Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates  

Here, we present an adaption of the chromosome conformation capture (3C) technique in detail with an emphasis on undergraduate involvement and learning.

Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C ...

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3.1K Views.  Bryant University. Here, we present an adaption of the chromosome conformation capture (3C) technique in detail with an emphasis on undergraduate involvement and learning.

Video: Author Spotlight: Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates  

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...