This work was in part supported by NCI (CA193549) and CCHMC Research Innovation Pilot awards to Kakajan Komurov, and Department of Defense (BC150484) award to Navneet Singh. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the Department of Defense. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The Institutional Animal Care and Use Committee and Institutional Biosafety Committee of the Cincinnati Children&#8217;s Research Foundation approved all animal experimental procedures (IACUC protocol #2017-0061 and IBC protocol #IBC2016-0016), and these experiments were carried out in accordance with standards as described in the NIH Guide to the Care and Use of Laboratory Animals.
1. Chronic inhibition of RNA Pol II by flavopiridol treatment&#8212;basic strategy
<ol>
	<li>Seed B16/F10 mouse melanoma cells in low density (0.2 x 106) in a 10 cm culture plate in their corresponding medium (Dulbecco&#8217;s Modified Eagle Medium [DMEM], 10% fetal bovine serum [FBS], 1% penicillin and streptomycin [Pen/Strep]) and incubate overnight in a 37 &#176;C, 5% CO2 humidified incubator.</li>
	<li>Following day, wash the cells with 1x phosphate-buffered saline (PBS) and add a new batch of culture media with a sublethal dose (estimated as 25&#8201;nM) of flavopiridol&#8212;an inhibitor of the RNA Pol II elongation factor p-TEFb (cyclin T/CDK9)&#8212;for one week without further sub-culturing.</li>
	<li>Following a week of flavopiridol treatment, perform confirmatory assays to evaluate the model&#8217;s ability to recapitulate various attributes of transcriptional elongation defects seen in TEdeff cancers.</li>
</ol>
2. Confirmatory immunoblot assay to assess defective RNA Pol II function and impairment of interferon (IFN) pathway and tumor necrosis factor (TNF) pathway signaling of in the generated mouse TEdeff model
<ol>
	<li>Culture equal number (105) of flavopiridol treated B16/F10 mouse melanoma cells and parental B16/F10 mouse melanoma cells in two different sets of 12 well plates (one set for RNA Pol II functional characterization and the other set for cytokine stimulation) at 37 &#176;C in a 5% CO2 humidified incubator overnight.</li>
	<li>Next day, treat the cells in the cytokine stimulation set with mouse IFN-&#947;, IFN-&#945; (5&#8201;ng/mL), or TNF-&#945; (5&#8201;ng/mL) for 45&#8201;min at 37 &#176;C.</li>
	<li>Now, extract protein from cells in both cytokine and RNA Pol II functional characterization sets using a radioimmunoprecipitation assay (RIPA) lysis buffer in the following manner:
	<ol>
		<li>Wash cells with 1x PBS and lyse it with 50 &#181;L of the lysis buffer per well. Scrape, and then pellet the lysed cells at 4 &#176;C, 21,130 x g.</li>
		<li>Measure the protein in cell lysate supernatants using a standard colorimetric assay for protein concentration following detergent solubilization (Bradford or a similar assay).</li>
	</ol>
	</li>
	<li>Load equal amount of measured protein (15&#8201;&#181;g) from each sample to run in a 4%&#8722;18% sodium dodecyl sulfate (SDS) polyacrylamide gel, and transfer them onto polyvinylidene difluoride (PVDF) membranes.</li>
	<li>Block the PVDF membranes in 5% dry milk in tris-buffered saline&#8722;polysorbate 20 (TBST) for 1&#8201;h followed by an overnight incubation at 4 &#176;C with primary antibodies (RNA Pol II 1:1000; p-SER2 RNA Pol II 1:1000; p-SER5 RNA Pol II 1:1000; H3K36me3 1:2000; total H3 1:2000; STAT1 1:1000; p-STAT1 1:1000; NF&#954;B 1:1000; p-NF&#954;B 1:1000; &#946;-Actin 1:5000) in 5% bovine serum albumin.</li>
	<li>Following day, wash the PVDF membranes with 1x TBST for 15 min at room temperature (RT), and incubate them with appropriate secondary antibodies (anti-rat [1:5000] for RNA Pol II, p-SER2 RNA Pol II, and p-SER5 RNA Pol II; anti-rabbit (1:5000) for H3K36me3, total H3, STAT1, p-STAT1, NF&#954;B, and p-NF&#954;B) for 50 min at RT. Detect the protein signals using commercially available horseradish peroxidase (HRP) substrate with enhanced chemiluminescence. 
	NOTE: &#946;-Actin primary used is HRP-conjugated, therefore it can be developed without a secondary.</li>
</ol>
3. Confirmatory assay to assess mRNA processing defects in the generated mouse TEdeff model
<ol>
	<li>Seed equal number (0.2 x 106) of flavopiridol treated B16/F10 mouse melanoma cells and parental B16/F10 mouse melanoma cells in 6-well plates at 37 &#176;C in a 5% CO2 humidified incubator overnight.</li>
	<li>Extract total RNA from the cultured cells at 60% confluency using an RNA extraction reagent or kit (Table of Materials).</li>
	<li>Deplete rRNA from the total extracted RNA in the following manner: 
	NOTE: A low-input protocol has been co-opted from a commercially available kit to deplete rRNA
	<ol>
		<li>Set one water bath or heat block to 70&#8722;75 &#176;C, and another water bath or heat block at 37 &#176;C.</li>
		<li>Add the total RNA (100&#8722;500 ng in 2 &#181;L of nuclease-free water) with 1 &#181;L of selective rRNA depletion probe and 30 &#181;L of hybridization buffer in a microcentrifuge tube, mix gently by vortexing and incubate them at 70&#8722;75 &#176;C for 5 min.</li>
		<li>Now, transfer the tubes to a 37 &#176;C water bath/heat block, and allow the sample to cool to 37 &#176;C over a period of 30 min.</li>
		<li>Resuspend the selective rRNA depletion probe magnetic beads by vortexing, and aliquot 75 &#181;L of beads in a 1.5 mL RNase-free microcentrifuge tube.</li>
		<li>Place the bead suspension on a magnetic separator for 1 min. Allow the beads to settle. Gently aspirate and discard the supernatant. Repeat washing the beads once again by adding 75 &#181;L of nuclease-free water and discarding the supernatant following magnetic separation.</li>
		<li>Resuspend the washed beads in 75 &#181;L of hybridization buffer, and aliquot 25 &#181;L of it to another tube and maintain it at 37 &#176;C for later use.</li>
		<li>Place the remaining 50 &#181;L beads on a magnetic separator for 1 min and discard the supernatant. Resuspend the beads in 20 &#181;L of hybridization buffer and maintain it at 37 &#176;C for later use.</li>
		<li>After the cooling of the RNA/selective rRNA depletion probe mixture to 37 &#176;C for 30 min, briefly centrifuge the tube to collect the sample to the bottom of the tube.</li>
		<li>Transfer 33 &#181;L of the RNA/selective rRNA depletion probe mixture to the prepared magnetic beads from step 3.3.7. Mix by low speed vortexing.</li>
		<li>Incubate the tube at 37 &#176;C for 15 min. During incubation, gently mix the contents occasionally. Followed by brief centrifugation to collect the sample to the bottom of the tube.</li>
		<li>Place the tube on a magnetic separator for 1 min to pellet the rRNA-probe complex. This time do not discard the supernatant. The supernatant contains rRNA-depleted RNA.</li>
		<li>Place the tube of 25 &#956;L of beads from step 3.3.6 on a magnetic separator for 1 min. Aspirate and discard the supernatant. Add the supernatant from step 3.3.11 to the new tube of beads. Mix by low speed vortexing.</li>
		<li>Incubate the tube at 37 &#176;C for 15 min. During incubation, gently mix the contents occasionally. Briefly centrifuge the tube to collect the sample to the bottom of the tube.</li>
		<li>Place the tube on a magnetic separator for 1 min to pellet the rRNA-probe complex. Do not discard the supernatant. Transfer the supernatant (about 53 &#956;L) containing rRNA-depleted RNA to a new tube.</li>
		<li>Measure the concentration of the RNA yield by a spectrophotometer.</li>
	</ol>
	</li>
	<li>Use one half of the rRNA-depleted samples as input to magnetic beads containing oligo (dT)25 to extract polyA+&#8201;RNA in the following manner: 
	NOTE: This protocol of isolating polyA tail messenger RNA using oligo dT sequences bound to the surface of magnetic beads has been co-opted from a commercially available kit (Table of Materials).
	<ol>
		<li>Resuspend the oligo dT beads in the vial by briefly vortexing for &#62;30 s and transfer 200 &#181;L of oligo dT beads to a tube. Add the same volume (200 &#181;L) of binding buffer, and resuspend.</li>
		<li>Place the tube in a magnet for 1 min and discard the supernatant. Now, remove the tube from the magnet and resuspend the washed oligo dT beads in 100 &#181;L of binding buffer.</li>
		<li>Adjust the volume of the input rRNA-depleted total RNA sample to 100 &#956;L with 10 mM Tris-HCl pH 7.5. Now, add 100 &#956;L of binding buffer.</li>
		<li>Heat to 65 &#176;C for 2 min to disrupt secondary RNA structures. Now, immediately place on ice.</li>
		<li>Add the 200 &#956;L of total RNA to the 100 &#956;L washed beads. Mix thoroughly and allow binding by rotating continuously on a rotor for 5 min at RT.</li>
		<li>Place the tube on the magnet for 1-2 min and carefully remove all the supernatant and carefully remove all supernatant.</li>
		<li>Remove the tube from the magnet and add 200 &#956;L of Washing Buffer.</li>
	</ol>
	</li>
	<li>Measure the purity and concentration of the extracted polyA+ RNA by a spectrophotometer. 
	NOTE: A 260/280 ratio of 1.90&#8722;2.00, and a 260/230 ratio of 2.00&#8722;2.20 for all RNA samples are considered acceptable.</li>
	<li>Use the remaining half of the rRNA-depleted samples from section 3.3 as input to protein A columns (provided in the RNA immunoprecipitation [RIP] kit, Table of Materials) to immunoprecipitate five-prime capped RNAs using monoclonal 7-methylguanosine antibody in the following manner: 
	NOTE: This protocol of isolating m7G capped messenger RNA using a commercially available RNA immunoprecipitation kit has been co-opted and further modified.
	<ol>
		<li>Wash the protein A magnetic beads obtained from the RIP kit according to manufacturer&#8217;s protocol to pre-bind the antibody to the beads.</li>
		<li>Transfer 3 &#181;g of 7-methylguanosine antibody (rabbit IgG provided in the kit can be used the negative control) to the beads in a microcentrifuge tube suspended in 100 &#181;L wash buffer from the kit.</li>
		<li>Incubate with low speed rotation for 30 min at RT. Centrifuge tubes briefly and then place the tubes on a magnetic separator, remove and discard the supernatant.</li>
		<li>Remove tubes and add 500 &#181;L of wash buffer from the kit and vortex briefly. Centrifuge the tubes briefly followed by magnetic separation once again, remove and discard the supernatant.</li>
		<li>Repeat step 3.6.4 once again.</li>
		<li>Add around 120 ng of rRNA depleted (from section 3.3) to the prewashed 7-methylguanosine antibody bound beads. Add 1 &#181;L of RNase inhibitor. Incubate at RT for 1&#8722;1.5 h with mild agitation.</li>
		<li>Spin down the beads at 300 x g for 10 s and remove the supernatant containing uncapped (non-7-methylguanosine) mRNA to a new microcentrifuge tube.</li>
		<li>Add 100 &#181;L of wash buffer and wash it two more times similarly. Pool the collected supernatant in the same microcentrifuge tube labelled uncapped (non-7-methylguanosine) mRNA. Store on ice.</li>
		<li>Elute the capped (7-methylguanosine) mRNA from the beads with 300 &#181;L of urea lysis buffer (ULB) containing 7 M urea, 2% SDS, 0.35 M NaCl, 10 mM EDTA and 10 mM Tris, pH 7.5 by heating the beads at 65 &#176;C for 2&#8722;3 min.</li>
		<li>Mix 300 &#181;L of the eluted samples (capped and uncapped mRNA) with 300 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1; commercially available) (stored at 4 &#176;C). Mix well by inverting and leave for about 10 min then mix again gently.</li>
		<li>Centrifuge at 18,928 x g for 2 min and carefully pipette the top layer to fresh tube and discard bottom layer.</li>
		<li>Add 300 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1; stored at 4 &#176;C) to the samples. Mix well by inverting and then centrifuge at 18,928 x g for 1 min. Carefully, pipette the top layer to a fresh tube and discard bottom layer.</li>
		<li>Add 300 &#181;L of 2-porpanol and 30 &#181;L of 3 M sodium acetate (pH 5.2) to the capped and uncapped RNA. Invert the sample a few times and put it at -20 &#176;C for 20 h.</li>
		<li>Now, centrifuge the samples at 18,928 x g for 10 min at 4 &#176;C. Carefully remove the supernatant and add 500 &#181;L of 70% ethanol.</li>
		<li>Centrifuge again at 18,928 x g for 10 min at 4 &#176;C. Carefully discard the supernatant and dry the pellet at RT for less than 5 min. Resuspend the pellet in nuclease-free water.</li>
	</ol>
	</li>
	<li>Measure the purity and concentration of RNA yield by a spectrophotometer. The 260/280 ratio should be in the range of 1.90&#8722;2.00, and the 260/230 ratio in the range of 2.00&#8722;2.20 for all RNA samples.</li>
</ol>
4. Confirmatory assay to assess the response of mouse TEdeff model to FasL mediated cell death
<ol>
	<li>Seed equal number (30,000 cells) of flavopiridol treated B16/F10 mouse melanoma cells and parental B16/F10 mouse melanoma cells in a 96-well culture plate in their corresponding medium (DMEM), and incubate overnight in a 37 &#176;C, 5% CO2 humidified incubator.</li>
	<li>Treat the cells in a culture hood with different concentrations of hhis6FasL (0.1&#8722;1000&#8201;ng/mL) in the presence of 10&#8201;&#956;g/mL anti-His antibody and incubate for 24&#8201;h at 37 &#176;C, 5% CO2 humidified incubator.</li>
	<li>Remove dead cells by washing with 1x PBS buffer. Fix the attached cells in 4% paraformaldehyde for 20 min at RT. Discard the 4% paraformaldehyde (no need to wash), and stain with crystal violet solution (20% methanol, 0.5% crystal violet in 1x PBS) for 30&#8201;min.</li>
	<li>Remove excess stain by gently rinsing the plates in tap water. Keep the plates to dry at RT.</li>
	<li>Re-dissolve the crystal violet in 100 &#181;L of 1x nonionic surfactant dissolved in 1x PBS, and measure cell density by measuring the absorbance at 570&#8201;nm in a microplate reader.</li>
</ol>
5. Exploratory assay to assess the response of mouse TEdeff model to antigen specific cytotoxic T-cell attack
<ol>
	<li>Isolation and activation of OT-I CD8+ cytotoxic T-cell attack (CTL)
	<ol>
		<li>Purify CD8+ cells from spleens of OT-I TCR Tg RAG-1&#8722;/&#8722; mice by magnetic cell separation using a mouse CD8 T cell isolation kit as follows:
		<ol>
			<li>Harvest two spleens from two OT-I TCR Tg RAG-1&#8722;/&#8722; mice in complete RPMI media.</li>
			<li>Mash the spleens in a 70 &#181;m filter kept on a 50 mL tube filled with 20 mL of RPMI, using the back of a syringe till only fat is left behind in the filter.</li>
			<li>Centrifuge the flow through at 220 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
			<li>Add 1 mL of red blood cell (RBC) lysis buffer to the spleen pellet from the previous centrifugation step and pipette the mixture for 1 min.</li>
			<li>Neutralize the solution by adding up to 10 mL of RPMI.</li>
			<li>Centrifuge at 220 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend in 10 mL of RPMI.</li>
			<li>Take a small aliquot for counting. Centrifuge the remaining at 220 x g for 5 min at 4 &#176;C.</li>
			<li>For every million cells counted, resuspend the pellet in 1 mL of commercially available magnetic separation system buffer (Mojo buffer or a similar buffer).</li>
			<li>Prepare an antibody cocktail of volume 100 &#181;L for every 1 mL of pelleted cells in step 5.1.1.8. The antibody cocktail includes: biotin anti-CD4, CD105, CD45R/B220, CD11c, CD49b, TER-119, CD19, CD11b, TCR &#947;/&#948;, and CD44.</li>
			<li>Add this cocktail to the 1 mL pelleted cells and keep on ice for 15 min.</li>
			<li>Add 100 &#181;L of magnetic (streptavidin) beads to every 100 &#181;L of the antibody cocktail added to the 1 mL resuspended pelleted spleen cells. Keep on ice for 15 min.</li>
			<li>Add 7 mL of commercially available magnetic separation system buffer. Now, aliquot about 3&#8722;4 mL of the mixture to a fresh tube. Mix well and fix it to the magnet for 5 min.</li>
			<li>Decant the liquid (contains CD8+ cells) to a fresh tube on ice. Now, aliquot the remaining 3&#8722;4 mL of mixture from step 5.1.1.12 to the tube and fix it to the magnet for 5 min. Decant the liquid (second batch of CD8+ cells isolated) to the same tube containing the first batch of CD8+ cells kept on ice.</li>
		</ol>
		</li>
		<li>Seed engineered adherent fibroblast APC&#173;-(MEC.B7.SigOVA) line to express a specific ovalbumin (OVA)-derived, H-2Kb-restricted peptide epitope OVA257-264 (SIINFEKL), along with the co-stimulatory molecule B7.1, at 75,000 cells per well in 24-well plates, at 37 &#176;C, 5% CO2 humidified incubator. 
		NOTE: The adherent fibroblast used is a gift from Dr. Edith Janssen&#8217;s lab at CCHMC. The line was created originally in Dr. Stephen P. Schoenberger&#8217;s lab in La Jolla Institute for Allergy and Immunology6.</li>
		<li>After 24&#8201;h, wash the monolayer of APC once with Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM) commercially available with HEPES buffer, sodium pyruvate, L-glutamine and high glucose), and add 0.5&#8201;x&#8201;106 naive OT-I CD8+ cells (from step 5.1.1.13) in 2&#8201;mL of IMDM supplemented with 50&#8201;mM &#946;-ME, 2&#8201;mL EDTA, 4&#8201;mM L-glutamine and HEPES and 10% FBS.</li>
		<li>After 20&#8201;h, gently harvest the non-adherent OT-I cells (by collecting the media in the culture dish with floating OT-I cells and pelleting the cells at 191 x g for 2 min; count the viable OT-I cells) and transfer them for co-culture.</li>
	</ol>
	</li>
	<li>Co-culture of CD8+ cells with B16/F10-OVA cells
	<ol>
		<li>Seed OT-I-derived CD8+ cells at a ratio of 1:1 (300,000 cells each) in a co-culture with B16/F10 (lacking the antigen ovalbumin), untreated B16/F10-OVA, and B16/F10-OVA cells pre-treated with flavopiridol (25 nM) for 1 week, in 6-well dishes with complete DMEM media for 20&#8201;h at 37 &#176;C in a 5% CO2 humidified incubator.</li>
		<li>After 20 h, remove the OT-I-derived CD8+ cells (by collecting the media in the culture dish with floating OT-I derived CD8+ cells). Wash the adherent B16/F10-OVA cells in 1x PBS.</li>
		<li>Trypsinize the three groups of attached B16/F10-OVA cells in 0.05% EDTA containing trypsin for 5 min. Pellet the trypsinized cells at 191 x g for 5 min.</li>
		<li>Stain the harvested B16/F10-OVA cells by incubating them in cold PBS (containing 0.5% FBS and 0.05% sodium azide) with viability dye and relevant labeled antibodies (fixable viability staining dye e780, AF647-conjugated mouse CD8 and BV421-conjugated mouse CD45).</li>
		<li>Analyze the viability of the three groups of B16/F10-OVA cells by flow cytometry.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Adelman, K., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoter-proximal+pausing+of+RNA+polymerase+II:+emerging+roles+in+metazoans.">Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.</a> Nature Reviews Genetics. 13 (10), (2012).</li><li>Margaritis, T., Holstege, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poised+RNA+polymerase+II+gives+pause+for+thought.">Poised RNA polymerase II gives pause for thought.</a> Cell. 133 (4), 581-584 (2008).</li><li>Modur, V., 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=Defective+transcription+elongation+in+a+subset+of+cancers+confers+immunotherapy+resistance.">Defective transcription elongation in a subset of cancers confers immunotherapy resistance.</a> Nature Communications. 9 (1), 4410 (2018).</li><li>Hargreaves, D. C., Horng, T., Medzhitov, R. <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+inducible+gene+expression+by+signal-dependent+transcriptional+elongation.">Control of inducible gene expression by signal-dependent transcriptional elongation.</a> Cell. 138 (1), 129-145 (2009).</li><li>Adelman, K., 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=Immediate+mediators+of+the+inflammatory+response+are+poised+for+gene+activation+through+RNA+polymerase+II+stalling.">Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (43), 18207-18212 (2009).</li><li>van Stipdonk, M. J., Lemmens, E. E., Schoenberger, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+CTLs+Require+a+Single+Brief+Period+of+Antigenic+Stimulation+for+Clonal+Expansion+and+Differentiation.">Na&#239;ve CTLs Require a Single Brief Period of Antigenic Stimulation for Clonal Expansion and Differentiation.</a> Nature Immunology. 2 (5), 423-429 (2001).</li><li>Gilchrist, D. 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=Regulating+the+regulators:+the+pervasive+effects+of+Pol+II+pausing+on+stimulus-responsive+gene+networks.">Regulating the regulators: the pervasive effects of Pol II pausing on stimulus-responsive gene networks.</a> Genes &amp; Development. 26 (9), 933-944 (2012).</li><li>Danko, C. G., 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=Signaling+pathways+differentially+affect+RNA+polymerase+II+initiation,+pausing,+and+elongation+rate+in+cells.">Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells.</a> Molecular Cell. 50 (2), 212-222 (2013).</li><li>Nechaev, S., Adelman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pol+II+waiting+in+the+starting+gates:+Regulating+the+transition+from+transcription+initiation+into+productive+elongation.">Pol II waiting in the starting gates: Regulating the transition from transcription initiation into productive elongation.</a> Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms. 1809 (1), 34-45 (2011).</li><li>Zhou, M., 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=Tat+modifies+the+activity+of+CDK9+to+phosphorylate+serine+5+of+the+RNA+polymerase+II+carboxyl-terminal+domain+during+human+immunodeficiency+virus+type+1+transcription.">Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription.</a> Molecular and Cellular Biology. 20 (14), 5077-5086 (2000).</li><li>Palancade, B., Bensaude, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+RNA+polymerase+II+carboxyl&#8208;terminal+domain+(CTD)+phosphorylation.">Investigating RNA polymerase II carboxyl&#8208;terminal domain (CTD) phosphorylation.</a> European Journal of Biochemistry. 270 (19), 3859-3870 (2003).</li></ol>

The authors have nothing to disclose.

RNA Pol II elongation control has emerged as a decisive lever for regulating stimulus-responsive gene expression to the benefit of malignant cells5,7,8. Overcoming promoter-proximal pausing to elongation and subsequent mRNA production requires the kinase activity of P-TEFb9,10,11. Our model utilizes flavopiridol (25 nM), an inhibitor of the essential cyclin-dependent kinase CDK9, to mimic the defects observed during Pol II elongation in TEdeff cancers&#8212;a previously unknown phenotype in cancers discovered by our group previously3.
CDK9 kinase activity has long been known to be essential for phosphorylation of serine 2 residues in the CTD of the large subunit of Pol II. Critically, we have succeeded in optimizing flavopiridol treated chronic inhibition of CDK9 (25 nM for 1 week) in B16/F10 OVA such that, in addition to inhibiting CTD phosphorylation, 25 nM flavopiridol treatment for 1 week prevents proper post-transcriptional modifications of mRNA in an unanticipated way and effectively abrogates p-TEFb-dependent productive elongation along long genes such as pro-inflammatory response signaling genes, significantly altering their patterns of expression both at the mRNA and protein levels. To the best of our knowledge, there is no other model described in literature which effectively achieves the same.
This easy to establish, generalizable model of TEdeff can therefore be leveraged to dissect, both transcriptional and epigenetic modifications enabling TEdeff cancers to adapt to immune-mediated cell attack. Moreover, this murine model retains its TEdeff-like reduction of total and phospho- RNA Pol II levels 21 days after flavopiridol release in in vivo growth assay3 (not mentioned in the protocol here), suggesting the extent of stability of this non-genetic model for further in vivo experimentation. However, care must be taken to optimize the exact sublethal dose of flavopiridol for other murine lines (e.g., about 20 nM flavopiridol treatment for 1 week is the sublethal dose for MC38 murine carcinoma line; not used in this protocol), the impact of variation in cell plating density, culture conditions, and cytokine stimulation conditions may vary for different murine lines. The protocol described here gives a basic framework to minimize the variables known to be critical for the generation of TEdeff-like features by chronic CDK9 inhibition. In addition, human carcinoma cell lines, such as T47D and CAL51 have been tested with short-term (3 days) flavopiridol treatments giving rise to similar TEdeff-like RNA Pol II profiles, indicating the usefulness of flavopiridol based chronic inhibition of CDK9 mediated transcription elongation in creating even model human lines to study TEdeff.

A key rate-limiting step in the expression of nearly all active genes is the transition of RNA polymerase II (RNA Pol II) from promoter-proximal pausing to productive elongation1,2. Given that epigenetic dysregulation of transcriptional elongation assists in the progression of multiple human malignancies defined as TEdeff, leading to suboptimal signaling in the pro-inflammatory response pathways amounting to a poor response and outcome to immunotherapy3, the overarching goal of this protocol is to establish a useful in vitro model to study these widespread non-genetic transcriptional abnormalities in cancers. In this light, the use of chronic pharmacological inhibition of CDK9 is an efficacious strategy for creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects. The rationale behind using chronic CDK9 inhibition is that it abrogates phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA Pol II, thus repressing the release of RNA Pol II into productive transcription elongation. Also, TEdeff cancers, described previously by our group3, are not classified under any specific somatic mutation. Therefore, a non-genetic (pharmacological) model is advantageous and best mimics the widespread transcriptional and epigenetic defects observed in them. The method herein details the generation and characterization of chronic flavopiridol treatment model of murine cancer cells. This method demonstrably disrupts transcription elongation along genes characterized by longer genomic lengths, with poised promoters and inducible expressions such as TNF/NF-&#954;B and interferon/STAT signaling, profoundly controlled at the level of transcription elongation3,4,5. Overall, this optimized murine cell line model of transcriptional elongation defects&#8212;the only model to our knowledge to study the newly described TEdeff tumors&#8212;drives resistance to anti-tumor immune attack, rendering a useful system to exploit and examine the vulnerabilities of non-genetic defects in core transcription machinery in cancers vis-&#224;-vis immune-mediated cell attack.

<table>
	<tbody>
		<tr>
			<td>hhis6FasL</td>
			<td>Cell Signaling</td>
			<td>5452</td>
			<td></td>
		</tr>
		<tr>
			<td>10X TBS</td>
			<td>Bio-Rad</td>
			<td>170-6435</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>20% methanol</td>
			<td>Fisher Chemical</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Falcon</td>
			<td>351147</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8211;18% SDS polyacrylamide gel</td>
			<td>Bio-Rad</td>
			<td>4561086</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr>
			<td>5% dry milk</td>
			<td>Bio-Rad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>7-Methylguanosine antibody</td>
			<td>BioVision</td>
			<td>6655-30T</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Cellstar</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>AF647-conjugated mouse CD8</td>
			<td>Biolegend</td>
			<td>100727</td>
			<td></td>
		</tr>
		<tr>
			<td>antibiotic and antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-His antibody</td>
			<td>Cell Signaling</td>
			<td>2366&#8201;P</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabit</td>
			<td>Cell Signaling</td>
			<td>7074</td>
			<td>Dilution 1:5000</td>
		</tr>
		<tr>
			<td>Anti-Rat</td>
			<td>Cell Signaling</td>
			<td>7077S</td>
			<td>Dilution 1:5000</td>
		</tr>
		<tr>
			<td>Bradford assay Kit</td>
			<td>Bio-Rad</td>
			<td>5000121</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ACROS Organics</td>
			<td>24040-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>BV421-conjugated mouse CD45</td>
			<td>Biolegend</td>
			<td>109831</td>
			<td></td>
		</tr>
		<tr>
			<td>crystal violet</td>
			<td>Sigma</td>
			<td>C3886-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Oligo (dT)25</td>
			<td>Ambion</td>
			<td>61002</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>45015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Live/Dead staining dye e780</td>
			<td>eBioscience</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Flavopiridol</td>
			<td>Selleckchem</td>
			<td>S1230</td>
			<td></td>
		</tr>
		<tr>
			<td>H3k36me3</td>
			<td>Abcam</td>
			<td>ab9050</td>
			<td>Dilution 1:2000</td>
		</tr>
		<tr>
			<td>IFN-&#945;</td>
			<td>R&#38;D systems</td>
			<td>12100-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>R&#38;D systems</td>
			<td>485-MI-100</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon Western Chemiluminescent HRP Substrate</td>
			<td>Millipore</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td></td>
		</tr>
		<tr>
			<td>NF-&#954;B</td>
			<td>Cell Signaling</td>
			<td>8242s</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>p-NF-&#954;B</td>
			<td>Cell Signaling</td>
			<td>3033s</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>p-Ser2-RNAPII</td>
			<td>Active Motif</td>
			<td>61083</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>p-Ser5-RNAPII</td>
			<td>Active Motif</td>
			<td>61085</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>p-STAT1</td>
			<td>Cell Signaling</td>
			<td>7649s</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>RiboMinu Eukaryote Kit</td>
			<td>Ambion</td>
			<td>A10837-08</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-24948</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAPII</td>
			<td>Active Motif</td>
			<td>61667</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>STAT1</td>
			<td>Cell Signaling</td>
			<td>9175s</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td>R&#38;D systems</td>
			<td>410-MT-010</td>
			<td></td>
		</tr>
		<tr>
			<td>total H3</td>
			<td>Cell Signaling</td>
			<td>4499</td>
			<td>Dilution 1:2000</td>
		</tr>
		<tr>
			<td>Tri reagent</td>
			<td>Sigma</td>
			<td>T9424</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>AA Hoefer</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Actin</td>
			<td>Cell Signaling</td>
			<td>12620S</td>
			<td>Dilution 1:5000</td>
		</tr>
		<tr>
			<td>&#946;-ME</td>
			<td>G Biosciences</td>
			<td>BC98</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine cell line based model of chronic cdk9 inhibition to study widespread non-genetic transcriptional elongation defects (tedeff) in cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

Here, we provide a detailed scheme (Figure 1) to establish a TEdeff cell model obtained by chronic sub-lethal (Figure 2) treatment with flavopiridol at 25 nM. In Figure 3, on 3 days of treatment with flavopiridol, B16 OVA cells show partial characteristics of TEdeff but after one week of treatment, B16/F10 OVA cells show a profound loss of phosphorylation at serine 2 position on the CTD of RNA Pol II along with a significant decrease in H3K36me3&#8212;a histone modification implicated in defining exon boundaries and an inhibitor of run-away cryptic transcriptions. As a consequence, TEdeff cell model shows critical mRNA processing defects with manifestly increased ratios of improperly capped and non-poly-adenylated mRNAs (Figure 4A,B). Also, specific repression of key inflammatory response pathway genes and FasL mediated cell death pathway are seen in Figure 5 and Figure 6. The imposed resistance to interferon (IFN-&#945;, IFN&#947;) and TNF-&#945; stimulated phosphorylation of STAT1 and NF&#954;B, and resistance to cell death by the death receptor ligand FasL drastically reduces the cytotoxicity of an immune cell attack against TEdeff tumors. These confirmatory techniques are designed to test the extent of influence chronic perturbation of transcription elongation has on a wide array of stimulus-responsive genes, and whether such a perturbation in a given mouse cell line model is adequate enough to prompt an acute dearth of functional mRNA in inflammatory response signaling genes, mimicking the basic essentialities of TEdeff cancers clinically. Based on our study of the flavopiridol treatment, the suppression of phosphorylation at the second serine residue (pSER2) of RNA Pol II CTD is critical, as it marks transcription elongation. A sublethal dose for any given mouse carcinoma cell line must achieve a reduction in pSER2 levels in addition to having an insignificant effect on the rate of growth and viability of the cell line. Although we consistently see a reduction in pSER2 and H3K36me3 levels on 25 nM flavopiridol treatments, it does not guarantee a repression of both pSTAT1 and pNF&#954;B levels (on IFN-&#945;, IFN&#947; and TNF-&#945; stimulations, respectively). Each mouse carcinoma cell line is unique (B16/F10 OVA or CT26 cells cultured in different labs over a period of time may have slightly altered effects) and they may have either JAK1 or CCNT1 partially rescuing the effects of flavopiridol in suppressing the inflammatory response pathway genes. In such cases, the kinetics of pSTAT1 and pNF&#954;B levels may need to be checked at different time points (5&#8722;70 min) to understand the temporality of flavopiridol mediated effects and its rescue by either JAK1 or CCNT1. Accordingly, JAK1 and/or CCNT1 may need to be knocked down to establish this model.
Once the flavopiridol model is established and characterized using the aforementioned assays, we provide an exploratory assay to test if the TEdeff cell model confers resistance to cytotoxic T-cell (CTL) attack. Based on our optimized protocol, flavopiridol treated B16/F10 cells stably overexpressing the OVA gene (B16 OVA) co-incubated with the activated CD8+&#8201;CTLs (specific for the OVA257-264 epitope) having selective toxicity to OVA-expressing cells (a gift from Dr. Stephen P. Schoenberger&#8217;s lab6) were not susceptible to OT-I CTL-mediated tumor lysis. B16/F10 OVA cells (not pretreated with flavopiridol) underwent massive cell death in this system, while B16/F10 parental cells survived, as they do not express OVA antigen (Figure 7). It is clear from the outcome of the suggested exploratory assay that chronic flavopiridol-induced TEdeff can bestow a means to escape from anti-tumor immune attack even in vivo. This can be further tested in in vivo tumor models to check the propensity of TEdeff models to escape innate and adaptive anti-tumor immune responses. Anti-asialo treatments could be used to regulate the activity of NK cells in vivo in tumor bearing mice. Also, immune checkpoint therapy (anti-CTLA4 and anti-PD1) can be administered to TEdeff tumor bearing mice.
In totality, the TEdeff confirmatory assays along with the suggested exploratory assay together demonstrate the utility of incorporating this TEdeff cell model in a whole host of other tumor-immune testing conditions. This model can help parse out the molecular details resulting from defective transcriptional elongation in tumor cells and their response to immune cell interactions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59910/59910fig1.jpg" /> 
Figure 1: Schematic representation of the work flow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig1large.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/59910/59910fig2.jpg" /> 
Figure 2: Cell growth characteristics of B16 OVA cells chronically treated with low-dose flavopiridol: Viability (measured by viability reagent) of control and flavopiridol-treated cells B16 OVA at indicated days post-treatment. This figure has been modified from Modur et al.3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig2large.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/59910/59910fig3.jpg" /> 
Figure 3: Confirmatory assay to assess RNA Pol II and histone profile: Immunoblots of indicated histone and RNA Pol II marks in B16 OVA cells treated with flavopiridol for 72&#8201;h or 1 week. This figure has been modified from Modur et al.3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig3large.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/59910/59910fig5.jpg" /> 
Figure 4: Confirmatory assay to assess severe defects in mRNA processing. Ratios of 5&#8242;-uncapped to 5&#8242;-capped (A) and 3&#8242;-non-polyadenylated to 3&#8242;-polyadenylated (B) mRNA concentrations after rRNA depletion in the indicated cell lines. Error bars represent standard deviation based on three technical replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig5large.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/59910/59910fig4.jpg" /> 
Figure 5: Confirmatory assay to assess cytokine stimulation profile. Immunoblots of STAT1, pSTAT1, NF&#954;B and p NF&#954;B in control and flavopiridol pre-treated B16 OVA cells stimulated with IFN-&#945;, IFN&#947; or TNF-&#945; for 30&#8201;min at (5&#8201;ng/mL). This figure has been modified from Modur et al.3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59910/59910fig6.jpg" /> 
Figure 6: Confirmatory assay to assess resistance to FasL mediated cell death in vitro. Control and flavopiridol pre-treated B16 OVA cells treated with FasL for 24 h readout measured by viability assay. This figure has been modified from Modur et al.3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59910/59910fig7.jpg" /> 
Figure 7: Exploratory assay to assess resistance of TEdeff model to antigen-restricted cytotoxic T cell mediated attack in vitro. Left: diagrammatic scheme of the exploratory assay. Right: relative viability of B16/F10-OVA cells co-cultured with activated CD8&#8201;+&#8201;CTLs (1:1 ratio) isolated from the spleens of OT-I mice. P: Welch two-sample t-test. This figure has been modified from Modur et al.3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59910/59910fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers at JoVE.com

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

a-murine-cell-line-based-model-chronic-cdk9-inhibition-to-study

Nanyang Technological University

Research

JoVE Journal

Genetics

5.5K Views.  Cincinnati Children's Hospital Medical Center (CCHMC). The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

Video: A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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 ...

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 ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers

Summary

Explore More Videos

Chapters in this video

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

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

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

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information