Name: genome-wide analysis using chip to identify isoform-specific gene targets
Uploaded: 2010-07-07T17:00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets at JoVE.com

This work was supported by 115347-RSG-08-271-01-GMC from ACS, and by CA138631 grant from NIH.

The protocol was originally adapted from B. Ren, 2001. It represents a slight modification of the protocol by Odom et al.5 that can be found at <a href="http://jura.wi.mit.edu/cgi-bin/young_public/navframe.cgi?s=22&amp;f=appendices_downloads">http://jura.wi.mit.edu/cgi-bin/young_public/navframe.cgi?s=22&amp;f=appendices_downloads</a>
1. Pre-block and binding of antibody to magnetic beads (Should be performed the night before next step)
<ol>
 <li> Wash 100 &mu;L of Dynabeads Protein G (per IP, combine for multiple IPs) in 1 mL of fresh BSA/PBS solution (50 mg BSA in 10 mL PBS- This solution will last for one week). </li>
 <li>Collect the beads using a magnetic stand and repeat the washing procedure two more times. </li>
 <li> Next, add 10 &mu;g of antibody to 250 &mu;l of the beads slurry in PBS/BSA solution (per IP) and incubate overnight on a rotating platform at 4&deg;C.</li>
 <li> Upon completion, wash the beads three times in 1 ml of the PBS/BSA solution and then resuspend in 10 &mu;l of the PBS/BSA solution (per IP).</li>
</ol>
2. Cell cross-linking
<ol>
 <li> To begin this procedure, grow approximately 108 cells for each immunoprecipitation, or IP. Here, diffuse histiocytic lymphoma U937 cells are used and induced for monocytic differentiation with TPA for 96 hours.</li>
 <li> Following cell growth, add formaldehyde solution directly to the media to a final concentration of 1%. Then, swirl the flasks briefly and allow them to sit at room temperature for 10 minutes. Then, aspirate the media and rinse the cells with 15 mL of ice-cold PBS. Repeat this wash once.</li>
 <li>Next, add 6 mL of Lysis Buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, containing protease inhibitors) to each of the flasks on ice. Then, rock the flasks for 20 minutes at 4&deg;C.</li>
 <li> Finally, harvest the cells using a cell scraper and transfer them to 15 mL conical tubes. At this point the cells can be stored at -80&deg;C.</li>
</ol>
3. Cell Sonication
<ol>
 <li>If the cells were frozen, thaw them out. Once thawed, spin the cells down at 3,000 rpm for 10 minutes at 4&deg;C and discard the supernatant. Then, resuspend the cells in 6 mL of Lysis Buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA, containing protease inhibitors). Rock the tubes gently at room temperature for 10 minutes.</li>
 <li> Repeat centrifugation and resuspend the cells in 2.5 mL of Lysis Buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, containing protease inhibitors).</li>
 <li> Next, prepare the suspension for sonication by placing it in a beaker of icy water with the microtip of a Branson 450 Sonifier set to between 50 and 60% amplitude.</li>
 <li> Sonicate the solution for a 30 second constant burst and cool on ice for 1 minute. Repeat these pulses 10 to 15 times. Then, pipette the contents of the tube up and down and transfer them to a new tube. Continue to pulse and cool the solution an additional 5 times.</li>
 <li> Following sonication, add 10% Triton X-100 to 1/10 of the solution volume. Transfer the lysates to 1.5 mL centrifuge tubes, and spin the debris out in a microcentrifuge. Then transfer the cell lysate to a new tube.</li>
</ol>
4. Chromatin Immunoprecipitation
<ol>
 <li>Prior to chromatin immunoprecipitation, or ChIP, save 50 &mu;L of the cell lysate as the input sample. Then, combine the cleared cell lysate with Dynabeads pre-bound to antibody that have been previously prepared as described above. Rock the mixture, in addition to the 50 &mu;L input sample, at 4&deg;C overnight.</li>
 <li>Wash the beads with 1 mL of the Wash Buffer (50 mM HEPES-KOH, pH 7.6, 0.5 M LiCl, 1mM EDTA, 0.7% sodium deoxycholate, 1% NP-40). Then, use a magnetic stand to collect the beads and remove the supernatant. Repeat this wash 6 to 8 times.</li>
 <li> Perform a final wash with 1 mL TE-plus-50 mM NaCl (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA). Spin the beads in a microcentrifuge at 3,000 rpm for 2 minutes at 4&deg;C. Following centrifugation, aspirate any residual TE buffer.</li>
 <li> Then, add 100 &mu;L of Elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) to the samples. Immediately after, incubate the samples at 65 &deg;C for 10 to 15 minutes. Scratch the tubes against a 1.5 mL tube rack every 2 minutes to keep the beads in the suspension.</li>
 <li> Collect the beads using centrifugation and the magnetic stand, and transfer the supernatant to a PCR tube. Then, retrieve the previously saved input sample and add 3 volumes of the Elution Buffer.</li>
 <li> Finally, place the IP and input samples into the thermal cycler overnight at 65 &deg;C to reverse the cross-linkages. </li>
 <li> The following day, add 1 volume of TE buffer to the samples. Then, add RNase A to a final concentration of 0.2 &mu;g/&mu;L. Incubate the samples in the thermal cycler for 1 to 2 hours at 37 &deg;C.</li>
 <li> After incubation, add proteinase K to a final concentration of 0.2 &mu;g/&mu;L. Then, incubate samples in the thermal cycler at 55 &deg;C for 2 hours. </li>
 <li> Next, extract the samples once with one volume of phenol. Extract the samples a second time with one volume of phenol:chloroform:isoamyl alcohol. Finally, extract the samples once more with one volume of chloroform:isoamyl alcohol.</li>
 <li> Then, add 30 &mu;g of glycogen to each sample. Also, add NaCl to a final concentration of 0.2 Molar and two volumes of ethanol to the samples. Incubate them for 30 minutes at -80 &deg;C. After incubation, spin the samples and decant the supernatant. Wash the pellets with 500 &mu;L of 75% ethanol. Then, dry the pellets and re-suspend them in 40 &mu;L of water. </li>
 <li> To check the sizes of the DNA fragments produced by the sonication procedure, load 5 &mu;g of the purified input sample in a 1.8% agarose gel and run the gel. The sonication procedure should produce fragments in the range of 150-350 base pairs. However, if the fragments do not fall in this range, the sonication procedure should be adjusted by varying the number of bursts and the amplitude.</li>
 <li> To check robustness and specificity of ChIP, the enrichment in control binding regions by performing gene-specific PCR with 1 &mu;l of the IP samples and a dilution series of the input sample. Two to three "bound" regions and an unbound control region need to be selected in order to test quality of IP and input samples.</li>
</ol>
5. Amplification of genomic DNA
<ol>
 <li> Starting with 34 &mu;l of the IP sample or 200 ng of the input sample, perform end repair, 'A' tail addition and ligation of adapters to DNA fragments using Genomic DNA Sample Prep Kit <a href="http://www.illumina.com/systems/genome_analyzer.ilmn">http://www.illumina.com/systems/genome_analyzer.ilmn</a> (Illumina, Inc., San Diego, CA). </li>
 <li> Purify the DNA using a MinElute PCR Purification Kit and then elute it in buffer EB (10 mM Tris Cl, pH 8.5) that has been pre-warmed to 50 &deg;C. For example, use 23 &mu;L and 46 &mu;L for the final elution of IP and input DNA samples, respectively.</li>
 <li> Make the PCR reaction using 23 &mu;L of DNA with adapters, and 25 &mu;L Phusion DNA polymerase from the kit, 1 &mu;L of 20 &mu;M Sol_PCR_1, and 1 &mu;L of 20 &mu;M Sol_PCR_2. Run the PCR reaction as follows: 1) 30 seconds at 98&deg;C; 2) 10 seconds at 98&deg;C; 3) 30 seconds at 65&deg;C; 4) 30 seconds at 72&deg;C; repeating steps step 2-4 18 times and then 5 minutes at 72&deg;C, finally holding at 4&deg;C.</li>
 <li> Following PCR amplification, purify the DNA with the QIAGEN MinElute PCR Purification Kit. Pre-warm 15 &mu;L of buffer EB to 50 &deg;C and elute the DNA. Dilute 0.5 &mu;L of the sample 1:4 and perform a Nanodrop reading.</li>
</ol>
6. Gel purification of amplified products
<ol>
 <li> To purify the amplified products, prepare a 1.8% agarose gel of 50 mL using HPLC-grade water. Add TAE and ethidium bromide after the agarose has melted. Then, pour the gel. Load the gel after adding 4 &mu;L of loading buffer to the input and IP samples. Then, run the gel at 120 volts for 45 minutes.</li>
 <li> Analyze the gel after it has run. The gel should reveal that fragments in the range between 150 and 350 base pairs are produced. They represent genomic DNA fragments between 50 and 250 base pairs in length and an adapter.</li>
 <li> Excise the region of gel containing the material in the 150 350 base pair range with a scalpel. Take care to avoid excising the adapter-adapter band, which runs at about 120 base pairs. Recover the DNA using a QIAquick Gel Extraction Kit per manufacturer's instructions. Specifically, use one column from the QIAquick Gel Extraction Kit for a gel slice of 400 mg or less. To begin the extraction process, add 3 volumes of QG buffer to 1 volume of gel. Add 1 volume of isopropanol and load the mixture on the column. Use 0.5 ml of QG to wash the column, followed by standard procedure described in kit manual. Use H2O pre-warmed to 50&deg;C to elute the DNA. Incubate the column with 30 &mu;l of H2O for 5 minutes at 37&deg;C before spinning it down. </li>
 <li> Dry the sample down to precisely 11 &mu;L in a Speedvac without heat. Remove 1 &mu;L of the sample and measure the DNA concentration using a Nanodrop spectrophotometer.</li>
 <li> Finally, identify enriched gene products using the Illumina Genome AnalyzerIIe as described at <a href="http://genesdev.cshlp.org/content/suppl/2008/12/15/22.24.3403.DC1/GuentherSuppMat.pdf">http://genesdev.cshlp.org/content/suppl/2008/12/15/22.24.3403.DC1/GuentherSuppMat.pdf</a> 6.</li>
</ol>
7. Representative Results
 <img src="/files/ftp_upload/2101/2101fig1.jpg" alt="Figure 1" /> 
 Figure 1. The overall goal of the following experiment is to identify genomic targets of KDM5A/JARID1A/RBP2 histone demethylase.
(P1) This is achieved by preparation of cross-linked chromatin that is sonicated to produce DNA fragments of appropriate size. (P2) As a second step, RBP2 bound DNA fragments are immunoprecipitated with RBP2 antibodies. (P3) Next, the recovered DNA fragments are repaired at the ends and adapters are ligated onto the ends of genomic DNA to prepare libraries of genomic DNA for analysis on the flow cells in the Illumina Cluster Station. The DNA libraries are amplified by PCR using low number of cycles. (P4) Finally, illumina sequenced short reads are uniquely aligned to the human genome, significant peaks are identified and annotated to the closest genes in order to identify RBP2 enriched regions. (P5) Results are obtained that show molecular functions associated with RBP2 target genes based on genome-wide identification of RBP2 enriched regions.
 <img src="/files/ftp_upload/2101/2101fig2.jpg" alt="Figure 2" /> 
 Figure 2. Checking the results of chromatin sonication To check the sizes of the DNA fragments produced by the sonication procedure, 5 &mu;g (1/10) of the purified input sample was loaded on 1.8% agarose gel. As expected, our sonication procedure produced fragments in the range of 150-350 bp. However, if the fragments do not fall in this range, the sonication procedure should be adjusted accordingly, by varying the number of bursts and the amplitude.
 <img src="/files/ftp_upload/2101/2101fig3.jpg" alt="Figure 3" /> 
 Figure 3. Gel purification of amplified products. The PCR products are run on a gel to remove the adapters and select a size-range of templates for the cluster generation platform. This gel shows that fragments in the range between 150 and 350 base pairs were produced. They represent genomic DNA fragments between 50 and 250 base pairs in length and an adapter. We excise the selected region of gel with a scalpel. Care should be taken to avoid the adapter-adapter band, which runs at about 120 base pairs.
 <img src="/files/ftp_upload/2101/2101fig4.jpg" alt="Figure 4" /> 
 Figure 4. Isoform-specific antibody allows distinguishing between large and small isoforms of RBP2. RBP2 protein structure is presented in domain view. RBP2 contains several domains: the catalytic histone demethylation JmjC domain and associated JmjN domain, an ARID domain capable of sequence-specific DNA binding, a C5HC2 zinc finger that may potentially interact with DNA or other proteins, and multiple PHD domains. The two anti-RBP2 antibodies that allow distinguishing between RBP2 isoforms were derived against the RBP2 fragments indicated by thick lines.
 <img src="/files/ftp_upload/2101/2101fig5a.jpg" alt="Figure 5a" /> 
 Figure 5a. Overview of RBP2 binding regions along with chromosome and Ensembl genes. Genomic coordinates of identified enriched RBP2 (all isoforms and large isoform) binding regions are presented chromosome wise. Occupances of Ensembl genes (version 54; hg18) in the chromosomes (chromosome 10 in this picture) are presented as black bars (top panel). Each RBP2 binding region is represented as a vertical line, where the middle panel shows all RBP2 isoforms binding regions on chromosome 10, and the bottom panel shows RBP2 large isoform binding regions. The middle and bottom panels give overview of overlapping and specific occupancy of RBP2 isoforms on chromosome 10.
 <img src="/files/ftp_upload/2101/2101fig5b.jpg" alt="Figure 5b" /> 
 Figure 5b. Functional enrichment analysis of RBP2 target genes. Heatmap showing FDR corrected significantly (p-value &le;0.05) enriched GO Molecular Function categories among the genes bound (closest genes to the RBP2 binding region) by RBP2 (all and large isoforms). Colors toward red indicate high statistic significance, yellow indicates low statistic significance, and gray indicates no statistic significance. Enrichment analysis shows the overlapping and isoform specific molecular functions of RBP2 target genes.

<ol>
	<li>Wang, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+malignancies+caused+by+dysregulation+of+a+chromatin-binding+PHD+finger.">Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger.</a> Nature. 459 (7248), (2009).</li><li>Benevolenskaya, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+pRB+to+the+PHD+protein+RBP2+promotes+cellular+differentiation.">Binding of pRB to the PHD protein RBP2 promotes cellular differentiation.</a> Mol Cell. 18 (6), 623-623 (2005).</li><li>Lopez-Bigas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+the+H3K4+histone+demethylase+RBP2+reveals+a+transcriptional+program+controlling+differentiation.">Genome-wide analysis of the H3K4 histone demethylase RBP2 reveals a transcriptional program controlling differentiation.</a> Mol Cell. 31 (4), 520-520 (2008).</li><li>Benevolenskaya, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3K4+demethylases+are+essential+in+development+and+differentiation.">Histone H3K4 demethylases are essential in development and differentiation.</a> Biochem Cell Biol. 85 (4), 435-435 (2007).</li><li>Odom, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+pancreas+and+liver+gene+expression+by+HNF+transcription+factors.">Control of pancreas and liver gene expression by HNF transcription factors.</a> Science. 303 (5662), 1378-1378 (2004).</li><li>Guenther, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+chromatin+at+genes+encoding+stem+cell+regulators+in+human+mixed-lineage+leukemia.">Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia.</a> Genes Dev. 22 (24), 3403-3403 (2008).</li></ol>

No conflicts of interest declared.

Functional differences between RBP2 isoforms have not been determined. We have used a comprehensive approach to identify genomic regions bound by RBP2 isoforms and define the functional categories that these regions represent. This was accomplished by ChIP-Seq analysis followed by bioinformatics analysis of obtained sequence reads.

RBP2 modifies methylated lysine residues on histone tails. We found that RBP2 large isoform containing the recognition module for methylated histone lysine and RBP2 small isoform lacking this module bind to different regions in the human genome (Figure 5A). Importantly, isoform-specific regions and overlapping regions belong to genes with different molecular functions (Figure 5B). For example, "chromatin binding" and "transcription factor binding" functions can be ascribed to gene targets of RBP2 small isoform but not of RBP2 large isoform (Figure 5B). By comparing actual gene sets generated for all isoforms and RBP2 large isoform (data not shown), we can also define if the large isoform is specifically recruited to certain genes.

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			Oligonucleotide sequences 2006 Illumina, Inc. All rights reserved.
<ul>
<li>Sol_PCR_1 
Sequence 5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-3'</li>
<li>Sol_PCR_2 
Sequence 5'-CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T-3'</li>
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genome-wide analysis using chip to identify isoform-specific gene targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Watch this Scientific Journal Video about Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets at JoVE.com

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).