Name: Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation (Video) | JoVE Journal
Uploaded: 2017-09-20T12:30:00.000+00:00
Duration: 11 min 6 s
Description: Learn how to induce B lymphocytes to undergo antibody class-switching and plasma cell differentiation using HDAC inhibitors. This method reveals epigenetic modulation of microRNAs and mRNAs in B cells.

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5 minutes

This work was supported by NIH grants AI 105813 and AI 079705 (to PC), the Alliance for Lupus Research Target Identification in Lupus Grant ALR 295955 (to PC), and the Arthritis National Research Foundation research grant (to HZ). TS was supported by the Pediatrics Medical Center, Second Xiangya Hospital, Central South University, Changsha, China, in the context of the Xiangya-UT School of Medicine San Antonio medical student visiting program.

The protocol follows the animal care guidelines of The Institutional Animal Care and Use Committees of the University of Texas Health Science Center at San Antonio.
1. Stimulation of Mouse B Cells for CSR, Plasma Cell Differentiation, and HDI Treatment
<ol>
	<li>Preparation of spleen cell suspensions 
	NOTE: Perform all steps in a laminar flow hood except for mouse euthanasia and dissection.
	<ol>
		<li>Euthanize the specific pathogen-free C57BL/6J mouse (8-12 weeks of age) by CO2 inhalation.</li>
		<li>Lay the mouse on dissecting board with the abdomen facing upward. Pin all four feet of the mouse with 18-gauge, 1.5-in hypodermic needles.</li>
		<li>Sterilize the skin using 70% ethanol-soaked wipes.</li>
		<li>Use one set of autoclaved surgical tools (forceps and dissecting scissors) to cut through the skin just below the ribcage to visualize the spleen (on the left side of the abdomen, just below the liver).</li>
		<li>Use another sterile forceps and scissors to extract the spleen, which lies beneath the greater curvature of the stomach, by clamping the spleen gently with forceps and cutting off all the connecting tissue with dissecting scissors. Make sure to remove all the connecting tissue.</li>
		<li>Place the spleen into a 1.5-mL microcentrifuge tube containing 1,000 &#181;L of complete RPMI1640 medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FBS), 2 mM L-glutamine, 100 &#181;g/mL penicillin, 100 &#181;g/mL streptomycin, 0.25 &#181;g/mL amphotericin B, and 50 &#181;M &#946;-mercaptoethanol).</li>
		<li>Place a 70-&#181;m cell strainer into a 50-mL polypropylene conical centrifuge tube and pour the spleen from the microcentrifuge tube onto the cell strainer. Use the tip of a 15 mL polypropylene conical centrifuge tube to gently mash the spleen through the strainer. Rinse the cell strainer with 15 to 20 mL of complete medium.</li>
		<li>Spin down the cells at 350 x g for 5 min and discard the supernatant.</li>
		<li>Remove the red blood cells from the spleen cell suspensions. Resuspend the cell pellet in ACK lysis buffer (5 mL per spleen). Incubate for 4 min at room temperature with occasional shaking and then quench with 25 mL of complete medium.</li>
		<li>Spin down the cells at 350 x g for 5 min and discard the supernatant. Resuspend the cell pellet in 1 or 10 mL of complete medium. Count the viable cells using a hemocytometer.</li>
	</ol>
	</li>
	<li>B-cell isolation 
	NOTE: Isolate B cells by negative selection following the manufacturer&#39;s instructions. Non-B cells are targeted for removal with biotinylated antibodies directed against non-B cells (CD4, CD8, CD11b, CD43, CD49b, CD90.2, Ly-6C/G (Gr-1), and TER119) and streptavidin-coated magnetic particles.
	<ol>
		<li>Prepare a 0.25&#160;to 2 mL cell suspension of 1 x 108 cells/mL in complete medium in a sterile 5 mL (12 x 75 mm) polystyrene round-bottom tube. Add normal rat serum (50 &#181;L/mL) to the sample.</li>
		<li>Add 50 &#181;L/mL isolation cocktail to the sample. Mix the cells by gently pipetting up and down 2 - 3 times and incubate at room temperature for 10 min.</li>
		<li>Add 75 &#181;L/mL streptavidin-coated magnetic particles to the sample. Mix the mixture by gently pipetting up and down 2-3 times and incubate for 2.5 min at room temperature.</li>
		<li>Add complete RPMI 1640 medium. Mix the cells by gently pipetting up and down 2 - 3 times.</li>
		<li>Place the tube (without the lid) into the magnet and incubate at room temperature for 2.5 min. Pour off the supernatant containing untouched B cells into a new 15-mL conical tube.</li>
		<li>Count the viable cells using a hemocytometer and Trypan Blue stain. Assess the purity of the B cells by flow cytometry analysis of B-cell surface markers, such as B220 and CD19.</li>
	</ol>
	</li>
	<li>B-cell stimulation and HDI treatment
	<ol>
		<li>Resuspend the purified B cells in complete RPMI 1640 medium at a final concentration of 0.3 x 106 cells/mL.</li>
		<li>Use a 48-well cell culture plate for cell culture. For each well, add 1 mL of purified B-cell suspension (0.3 x 106 cells/mL), LPS (3 &#181;g/mL final concentration) from Escherichia coli, IL-4 (5 ng/mL final concentration), and 0 or 500 &#181;M HDI (valproic acid; VPA).</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 for 60 h for qRT-PCR and high-throughput mRNA- and miRNA-sequencing analysis, or 96 h for flow cytometry analysis.</li>
	</ol>
	</li>
	<li>Flow cytometry analysis of CSR and plasma cell differentiation
	<ol>
		<li>After 96 h of culture, detach the cells from each well by pipetting the cells up and down several times. Transfer the cell suspension into a 1.5-mL microcentrifuge tube. Spin down the cells at 350 x g for 5 min using a bench-top centrifuge and discard the supernatant.</li>
		<li>Resuspend the cells with 100 &#181;L of HBSS buffer containing 1% BSA and 0.5 ng/mL fluorescein (FITC)-conjugated goat anti-mouse IgM antibody (Ab), 0.2 ng/mL allophycocyanin (APC)-conjugated rat anti-mouse IgG1 monoclonal Ab (mAb), 0.2 ng/mL phycoerythrin (PE)-conjugated rat anti-mouse B220 mAb, 0.2 ng/mL PE-Cy7-conjugated rat anti-mouse CD138 mAb, and 2 ng/mL 7-aminoactinomycin D (7-AAD).</li>
		<li>Incubate the cells with fluorescence-conjugated antibodies (step 1.4.2) in the dark at room temperature for 30 min.</li>
		<li>Wash the cells with 1 mL of HBSS with 1% BSA.</li>
		<li>Spin down the cells at 1,500 x g for 5 min using a benchtop centrifuge and discard the supernatant.</li>
		<li>Resuspend the cells in 300 &#181;L of HBSS with 1% BSA and transfer the cell suspension to a round-bottom polystyrene tube. Cover the tube with foil to avoid light exposure.</li>
		<li>Perform flow cytometry analysis on a single-cell suspension. Collect 50,000 events for each compensation sample and 250,000 events for the other samples. Analyze the data using equipment software.</li>
		<li>Eliminate the debris and doublets by using a pulse geometry gate (FSC-H x FSC-A and SSC-H x SSC-A). Appropriately gate the plot on 7-AAD to exclude dead cells.</li>
	</ol>
	</li>
</ol>
2. High-Throughput mRNA-Seq
<ol>
	<li>After 60 h of culture, extract the total RNA from 2 - 4 &#215; 106 cells using a total RNA isolation kit that can recover small RNA following the manufacturer&#39;s instructions. Include a DNase I treatment step.</li>
	<li>Verify the RNA integrity using a bioanalyzer, following the manufacturer&#39;s instructions.</li>
	<li>Use 500-1,000 ng of high-quality total RNA (RNA integrity number RIN &#62; 8.0) for RNA-seq library preparation with a commercial RNA sample prep kit following the manufacturer&#39;s instructions.</li>
	<li>Pool the individual mRNA-seq libraries based on their respective 6-bp index portions of the adapters and sequence the libraries at 50 bp/sequence. Use a high-throughput DNA system according to the manufacturer&#39;s protocols.</li>
	<li>After the sequencing run, demultiplex with CASAVA to generate the fastq file for each sample. Perform reads mapping and bioinformatics analysis, as previously outlined11.</li>
	<li>Align all sequencing reads with their reference genomes (UCSC mouse genome build mm9) using TopHat2 default settings14. Process the bam files from alignment using HTSeq-count to obtain the counts per gene in all samples.</li>
</ol>
3. High-Throughput miRNA-Seq
<ol>
	<li>Use 100 ng-1 &#181;g of high-quality total RNA, as prepared in step 2.1, for small RNA-seq library preparation by using a commercial small RNA-seq kit.</li>
	<li>Ligate the degenerated 3&#39; adapter onto the 5&#8242; ends of the starting small RNA molecules with a commercial ligation kit. Ligate the degenerated 5&#39; adapter onto the 3&#8242; ends of the starting small RNA molecules with a commercial ligation kit. Convert the RNA to cDNA by reverse transcription and amplify the small RNA-seq library by PCR amplification with commercial kits.</li>
	<li>Use a 6% TBE native PAGE gel to isolate the final small RNA-seq library. Run the gel with 1X TBE buffer at 200 V until the bromophenol blue tracking dye band nears the bottom of the gel (0.5 - 1 cm).</li>
	<li>Remove the gel from the glass plates and stain with ethidium bromide (0.5 &#181;g/mL in water) in a clean container for 2-3 min. Visualize the gel bands on a UV transilluminator or another gel documentation instrument.</li>
	<li>Cut out the ~150-bp band using a clean razor and place it into a 1.7 mL tube.</li>
	<li>Extract the DNA using a gel extraction kit per manufacturer instructions.</li>
	<li>Check the size distribution of the final library with a commercial high-sensitivity DNA assay and the concentration with a commercial dsDNA assay per the manufacturers&#39; instructions.</li>
	<li>Pool the libraries for amplification and a subsequent sequencing run with a commercial high-throughput DNA sequencing system per the manufacturer&#39;s protocols.</li>
	<li>Demultiplex with CASAVA to generate the fastq file for each sample per the manufacturer&#39;s protocols.</li>
	<li>For small RNA-seq analysis of each sample, use Flicker for small RNA alignment per the manufacturer&#39;s protocols.
	<ol>
		<li>Remove reads that are aligned to contaminants, such as mitochondria, rRNA, primers, and so on.</li>
		<li>Align the data to mature miRNA sequences.</li>
		<li>Align the data to hairpin loop sequences (precursor miRNA).</li>
		<li>Align the data to other small RNA sequences (using the fRNA database)15.</li>
	</ol>
	</li>
	<li>After all samples are quantified, define the differential expressed miRNAs between different groups/samples. Predict miRNAs that target selected mRNAs or mRNAs that can be targeted by a selective miRNA using online miRNA target prediction tools, such as TargetScan16, MicroCosm17, and PicTar18.</li>
	<li>If functional annotation or pathway analysis is needed, submit the predicted genes to Ingenuity Pathway analysis (IPA) or DAVID.</li>
</ol>
4. Quantitative RT-PCR (qRT-PCR) of mRNAs and miRNAs
<ol>
	<li>qRT-PCR analysis of mRNA
	<ol>
		<li>Extract RNA from 0.2-5 &#215; 106 cells using a commercial total RNA isolation kit that can recover small RNA, following the manufacturer&#39;s instructions. Include a DNase I treatment step.</li>
		<li>Synthesize cDNA from total RNA with a first-strand cDNA synthesis system using oligo-dT primer.</li>
		<li>Quantify cDNA by qRT-PCR with appropriate primers, using 2x real-time PCR master mix with the following protocol: 95 &#176;C for 15 s, 40 cycles of 94 &#176;C for 10 s, 60 &#176;C for 30 s, and 72 &#176;C for 30 s.</li>
		<li>Perform data acquisition during the 72 &#176;C extension step and perform melting curve analysis from 72 to 95 &#176;C.</li>
	</ol>
	</li>
	<li>qRT-PCR analysis of miRNA
	<ol>
		<li>Aliquot RNA from the samples prepared in step 4.1.1.</li>
		<li>Reverse-transcribe the RNA into cDNA. Use a commercial microRNA reverse transcription kit, following the manufacturer&#39;s instructions.</li>
		<li>Perform real-time PCR for microRNA using a SYBR Green real-time PCR master mix with 250 nM mature miRNA forward primers in conjunction with a universal reverse primer.
		<ol>
			<li>Thaw 2x PCR Master Mix, miRNA-specific forward primer, and universal reverse primer at room temperature. Mix the individual solutions.</li>
			<li>Prepare a reaction mix as follows for a 25 &#181;L-per-well reaction volume (used in 96-well PCR plates): 
			2x PCR Master Mix 12.5 &#181;L 
			miRNA forward primers (2.5 &#956;M) 2.5 &#181;L 
			Universal reverse primer (2.5 &#956;M) 2.5 &#181;L 
			RNase-free water5 &#181;L</li>
			<li>Add 22.5 &#956;L of reaction mix to a 96-well PCR plate. Add 2.5 &#181;L of template cDNA (50 pg-3 ng) to the individual plate wells.</li>
			<li>Tightly seal the plate film. Centrifuge the plate for 1 min at 1,000 x g and room temperature.</li>
			<li>Place the plate in the real-time cycler and start the following cycling program: 95 &#176;C for 5 min, 40 cycles of 94 &#176;C for 15 s, 55 &#176;C for 30 s, and 72 &#176;C for 30 s.</li>
		</ol>
		</li>
		<li>Use the &#916;&#916;Ct method for qRT-PCR data analysis with a spreadsheet. Normalize the expression of the relevant miRNAs to the expression of small nuclear/nucleolar RNAs Rnu6/RNU61/2, Snord61/SNORD61, Snord68/SNORD68, and Snord70/SNORD70.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Perform statistical analysis to determine the p values by paired and unpaired Student&#39;s t-test using a spreadsheet and consider p values &#60;0.05 as significant.</li>
	</ol>
	</li>
</ol>

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<li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNAs%3A+target+recognition+and+regulatory+functions%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li>
<li>Fabian, M. R., Sonenberg, N., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+mRNA+translation+and+stability+by+microRNAs%5BTitle%5D)+AND+%22Annu.+Rev.+Biochem%22%5BJournal%5D)">Regulation of mRNA translation and stability by microRNAs.</a> Annu. Rev. Biochem. 79, 351-379 (2010).</li>
<li>White, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+deacetylase+inhibitors+upregulate+B+cell+microRNAs+that+silence+AID+and+Blimp-1+expression+for+epigenetic+modulation+of+antibody+and+autoantibody+responses%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses.</a> J Immunol. 193, 5933-5950 (2014).</li>
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The authors declare that they have no competing financial interests.

This protocol provides comprehensive approaches to induce B cell class switching and plasma cell differentiation; to analyze their impact by epigenetic modulators, namely HDI; and to detect the effect of HDI on mRNA and miRNA expression in these cells. Most of these approaches can also be used to analyze the impact of epigenetic factor on human B-cell function and mRNA/miRNA expression. The qRT-PCR and mRNA-seq/miRNA-seq approaches can also be used to analyze B cells isolated from mice treated with epigenetic modulators,...

Epigenetic marks or factors, such as DNA methylation, histone posttranslational modifications, and non-coding RNAs (including microRNAs), modulate cell function by altering gene expression1. Epigenetic modifications regulate B lymphocyte function, such as immunoglobulin class-switch DNA recombination (CSR), somatic hypermutation (SHM), and differentiation to memory B cells or plasma cells, thereby modulating the antibody and autoantibody responses2,3. CSR and SHM critically require activation-induced cytidine deaminase (AID, encoded as Aicda), which is highly induced in B cells in response to T-dependent and T-independent antigens4. Class-switched/hypermutated B cells further differentiate into plasma cells, which secrete large volumes of antibodies in a fashion critically dependent upon B lymphocyte-induced maturation protein 1 (Blimp1, encoded as Prdm1)5. Abnormal epigenetic changes in B cells may result in aberrant antibody/autoantibody responses, which can lead to allergic response or autoimmunity1,4. Understanding how epigenetic factors, such as miRNAs, modulate B cell-intrinsic gene expression is not only important for vaccine development, but is also essential to reveal the mechanisms of potential abnormal antibody/autoantibody responses.
Histone acetylation and deacetylation are modifications of the lysine residues on histone proteins typically catalyzed by histone acetyltransferase (HAT) and histone deacetylase (HDAC). These modifications lead to the increasing or decreasing accessibility of chromatin and further allow or prevent the binding of transcription factors or proteins to DNA and the alteration of gene expression5,6,7,8. HDAC inhibitors (HDI) are a class of compounds that interfere with the function of HDACs. Here, we used HDI (VPA) to address the regulation of HDAC on the intrinsic gene expression profile of B cells and on its mechanism.
miRNAs are small, non-coding RNAs approximately 18 to 22 nucleotides in length that are generated through several stages. miRNA host genes are transcribed and form hairpin primary microRNAs (pri-miRNAs). They are exported to the cytoplasm, where pri-miRNAs are further processed into precursor miRNAs (pre-miRNAs). Finally, mature miRNAs are formed through the cleavage of the pre-miRNAs. miRNAs recognize the complementary sequences within the 3&#39; untranslated region of their target mRNAs6,7. Through post-transcriptional silencing, miRNAs regulate cellular activity, such as proliferation, differentiation, and apoptosis10,11. Since multiple miRNAs can target the same mRNA, and one single miRNA can potentially target multiple mRNAs, it is important to have an in-context view of the miRNA expression profile to understand the value of the individual and the collective effect of miRNAs. miRNAs have been shown to be involved in B-cell development and peripheral differentiation, as well as B-cell stage-specific differentiation, antibody response, and autoimmunity1,4,9. In the 3&#39; UTR of Aicda and Prdm1, there are several validated or predicted evolutionarily conserved sites that can be targeted by miRNAs8.
Epigenetic modulation, including histone post-transcription modification and miRNAs, display a cell-type and cell stage-specific regulation pattern of gene expression9. Here, we describe methods to define the HDI-mediated modulation of miRNA and mRNA expression, CSR, and plasma cell differentiation. These include protocols for inducing B cells to undergo CSR and plasma cell differentiation; for treating the B cells with HDI; and for analyzing miRNA and mRNA expression by qRT-PCR, miRNA-seq, and mRNA-seq10,11,12,8,13.

<table><tbody><tr><td>C57BL/6 mice</td><td>Jackson Labs</td><td>664</td><td></td></tr><tr><td>Corning cellgro RPMI 1640 Medium (Mod.) 1X with L-Glutamine (Size: 6 x 500mL; With L-Glutamine)</td><td>Fisher Scientific</td><td>MT 10-040-CV&amp;nbsp;</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH300</td><td></td></tr><tr><td>HyClone Antibiotic Antimycotic Solution 100 mL</td><td>Fisher Scientific - Hyclone</td><td>SV3007901&amp;nbsp;</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Fisher Scientific</td><td>44-420-3250ML</td><td></td></tr><tr><td>Falcon Cell Strainers</td><td>Fisher Scientific</td><td>21008-952&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Trypan Blue Stain 0.04%</td><td>GIBCO/Life Technologies/Inv</td><td>15250</td><td></td></tr><tr><td>ACK Lysis Buffer&amp;nbsp;</td><td>Fisher Scientific</td><td>BW10-548E&amp;nbsp;</td><td></td></tr><tr><td>Hausser Scientific Bright-Line Counting Chamber</td><td>Fisher Scientific</td><td>02-671-51B</td><td></td></tr><tr><td>EasySep Magnet</td><td>Stem Cell Technologies</td><td>18000</td><td></td></tr><tr><td>Falcon Round-Bottom Polystyrene Tubes with Cap</td><td>Fisher Scientific</td><td>14-959-1A</td><td></td></tr><tr><td>EasySep Mouse B cell Isolation Kit</td><td>Stem Cell Tech</td><td>19854</td><td></td></tr><tr><td>BD Needle Only 18 Gauge 1.5 inch SHORT BEVEL 100/box&amp;nbsp;</td><td>BD Biosciences</td><td>&amp;nbsp;305199</td><td></td></tr><tr><td>PE/Cy7 anti-mouse CD138 (Syndecan-1) Antibody</td><td>BioLegend&amp;nbsp;</td><td>142513 (25 ug)</td><td></td></tr><tr><td>PE-Cy7 B220 antibody</td><td>BioLegend</td><td>103222</td><td></td></tr><tr><td>7-AAD (1 mg)</td><td>Sigma Aldrich</td><td>A9400-1MG</td><td></td></tr><tr><td>APC anti-mouse/human CD45R/B220 antibody</td><td>Biolegend</td><td>103212</td><td></td></tr><tr><td>Mouse APC-IgG1 200 &amp;micro;g</td><td>Biolegend</td><td>406610</td><td></td></tr><tr><td>FITC anti-mouse IgM Antibody</td><td>Biolegend</td><td>406506</td><td></td></tr><tr><td>FITC anti-mouse/human CD45R/B220 Antibody</td><td>Biolegend</td><td>103206</td><td></td></tr><tr><td>PE Anti-Human/Mouse CD45R (B220) (RA3-6B2)</td><td>Biolegend</td><td>103208</td><td></td></tr><tr><td>HBSS 1X</td><td>Fisher Scientific</td><td>MT-21-022-CM</td><td></td></tr><tr><td>Bovine Serum Albumin, Fraction V, Heat Shock Treated&amp;nbsp;</td><td>Fisher Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>LPS 25mg (Lipopolysaccharides from Escherichia coli 055:B5)</td><td>Sigma Aldrich</td><td>L2880-25MG</td><td></td></tr><tr><td>Recombinant mouse IL-4 (carrier-free)</td><td>BioLegend</td><td>574302 (size: 10 ug)</td><td></td></tr><tr><td>Valproic acid sodium salt</td><td>Sigma Aldrich</td><td>P4543</td><td></td></tr><tr><td>SterilGARD e3 Class II Type A2 Biosafety Cabinet</td><td>The Baker Company</td><td>SG404</td><td></td></tr><tr><td>Large-Capacity Reach-In CO&lt;sub&gt;2&lt;/sub&gt; Incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>3950</td><td></td></tr><tr><td>Isotemp Digital-Control Water Baths: Model 205</td><td>Fisher Scientific</td><td>15-462-5Q</td><td></td></tr><tr><td>5mL Round Bottom Polystyrene Test Tube</td><td>Fisher Scientific</td><td>&amp;nbsp;14-959-5</td><td></td></tr><tr><td>Corning CentriStar 15ml Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>05-538-59A</td><td></td></tr><tr><td>1.7 mL Microtube, clear</td><td>Genesee</td><td>22-282</td><td></td></tr><tr><td>Higher-Speed Easy Reader Plastic Centrifuge Tubes 50ml</td><td>Fisher Scientific</td><td>06-443-18</td><td></td></tr><tr><td>ELMI SkySpin CM-6MT</td><td>ELMI</td><td>CM-6MT</td><td></td></tr><tr><td>Rotor 6M</td><td>ELMI</td><td>6M</td><td></td></tr><tr><td>Rotor 6M.06</td><td>ELMI</td><td>6M.06</td><td></td></tr><tr><td>Drummond Portable Pipet-Aid XP Pipet Controller</td><td>Drummond Scientific</td><td>4-000-101</td><td></td></tr><tr><td>25 mL serological pipette tips</td><td>Fisher Scientific</td><td>89130-900</td><td></td></tr><tr><td>10 mL serological pipette tips</td><td>Fisher Scientific</td><td>89130-898</td><td></td></tr><tr><td>5 mL serological pipette tips</td><td>Fisher Scientific</td><td>898130-896</td><td></td></tr><tr><td>48-well plates</td><td>Fisher Scientific</td><td>07-200-86</td><td></td></tr><tr><td>Allegra 6 Benchtop Centrifuge, Non-Refrigerated</td><td>Beckman Coulter</td><td>366802</td><td></td></tr><tr><td>GH-3.8A Rotor, Horizontal, ARIES Smart Balance</td><td>Beckman Coulter</td><td>366650</td><td></td></tr><tr><td>Allegra 25R Benchtop Centrifuge, Refrigerated</td><td>Beckman Coulter</td><td>369434</td><td></td></tr><tr><td>TA-15-1.5 Rotor, Fixed Angle</td><td>Beckman Coulter</td><td>368298</td><td></td></tr><tr><td>Fisher Scientific AccuSpin Micro 17</td><td>Fisher Scientific</td><td>13-100-675</td><td></td></tr><tr><td>Fisher Scientific Analog Vortex Mixer&amp;nbsp;</td><td>Fisher Scientific</td><td>02-215-365</td><td></td></tr><tr><td>miRNeasy Mini Kit (50)</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Direct-zol RNA MiniPrep kit</td><td>Zymo Research</td><td>R2050</td><td></td></tr><tr><td>Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)</td><td>Fisher Scientific</td><td>BP1145-1</td><td></td></tr><tr><td>Rnase-Free Dnase set (50)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>NanoDrop 2000 Spectrophotometers</td><td>Thermo Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Superscript III First-strand Synthesis System RT-PCR</td><td>Invitrogen</td><td>175013897</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-rad&amp;nbsp;</td><td>172-5121</td><td></td></tr><tr><td>Fisherbrand 96-Well Semi-Skirted PCR Plates, case of 25</td><td>Fisher</td><td>14-230-244</td><td></td></tr><tr><td>Microseal &#039;B&#039; Adhesive Seals</td><td>Bio-Rad</td><td>MSB-1001</td><td></td></tr><tr><td>MyiQ Optics Module</td><td>Bio-Rad</td><td>170-9744</td><td></td></tr><tr><td>iCycler Chassis</td><td>Bio-Rad</td><td>170-8701</td><td></td></tr><tr><td>Optical Kit</td><td>Bio-Rad</td><td>170-9752</td><td></td></tr><tr><td>BD LSR II Flow Cytometry Analyzer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FACSDiva software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo 10</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2943CA</td><td></td></tr><tr><td>S200 Focused-ultrasonicator</td><td>Covaris</td><td>S200</td><td></td></tr><tr><td>SPRIworks Fragment Library System I for Illumina</td><td>Beckman Coulter</td><td>A288267</td><td></td></tr><tr><td>cBot Cluster Generation Station</td><td>illumina</td><td>SY-312-2001</td><td></td></tr><tr><td>HiSeq 2000 Genome Sequencer</td><td>Illumina</td><td>SY-401-1001</td><td></td></tr><tr><td>TruSeq RNA Library Prep Kit v2</td><td>Illumina</td><td>RS-122-2001</td><td></td></tr><tr><td>TruSeq Small RNA Library Prep Kit</td><td>Illumina</td><td>RS-200-0012</td><td></td></tr><tr><td>NEXTflex Illumina Small RNA Sequencing Kit v3</td><td>Bioo Scientific</td><td>5132-05</td><td></td></tr><tr><td>2200 TapeStation</td><td>Agilent</td><td>G2964AA</td><td></td></tr></tbody></table>

genome-wide analysis of hdac inhibitor-mediated modulation of micrornas and mrnas in b cells induced to undergo class-switch dna recombination and plasma cell differentiation

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Using our protocol, purified B cells placed with LPS (3 µg/mL) and IL-4 (5 ng/mL) for 96 h can induce 30-40% of CSR to IgG1 and ~10% of plasma cell differentiation. After treatment with HDI (500µM VPA), the CSR to IgG1 decreased to 10-20%, while plasma cell differentiation decreased to ~2% (Figure 1). HDI-mediated inhibition of CSR was further confirmed by decreased numbers of post-recombination Iμ-Cγ1 and mature VHDJH-Cγ1 transcripts, which are the ...

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

The overall goal of this experiment is to induce B lymphocytes to undergo antibody class-switching and plasma cell differentiation and analyze the modulation as well as epigenetic regulation by the expression of HDAC inhibitor mRNA and microRNA in B-cells. This method can help answer key questions in the B-cell antibody response field such as, epigenetic modulation of immunoglobulin class-switching and plasma cell differentiation. The main advantage of this technique is to provide a simple method to modulate B-cell functions and to define the alteration of microRNAs and mRNAs by epigenetic regulators.After euthanizing a specific pathogen-free C57BL6J mouse and sterilizing the skin according to the text protocol, use autoclaved forceps and dissecting scissors to cut through the skin just below the ribcage to visualize the spleen on the left side of the abdomen, just below the liver. Gently clamp the spleen and cut off all the connecting tissue before extracting the organ. Then place the spleen into a 1.5 milliliter microcentrifuge tube containing 1, 000 microliters of complete RPMI 1640 medium.Next, insert a 70 micron cell strainer into a 50 milliliter polypropylene conical centrifuge tube and pour the spleen from the microcentrifuge tube onto the cell strainer. Use the tip of a 15 milliliter polypropylene conical centrifuge tube to gently mesh the spleen through the strainer. Then with 15 to 20 milliliters of complete medium, rinse the strainer.Spin down the cells at 350 x G for five minutes. Discard the supernatant. Use one milliliter of ACK lysis buffer to re-suspend the cell pellet and incubate for one minute.Then add four milliliters of ACK and incubate the suspension to remove red blood cells from the spleen suspension for an additional four minutes at room temperature with occasional shaking. Then add 25 milliliters of complete medium to quench the lysis reaction. Spin down the cells at 350 x G for five minutes.Discard the supernatant and add one milliliters of complete medium to re-suspend the cell pellet. Then using a hemocytometer, count the viable cells. In a sterile five milliliter polystyrene round bottom tube, prepare a 0.25 to two milliliter cell suspension of 1 x 10 to the 8th cells per milliliter in complete medium.Add normal rat serum to the sample. Next, add 50 microliters per milliliter of isolation cocktail to the tube. Then mix the cells by gently pipetting up and down two to three times and incubate the sample at room temperature for 10 minutes.Add 75 microliters per milliliter of streptavidin encoated magnetic particles to the tube. Gently pipette up and down two to three times and incubate the sample at room temperature for two and a half minutes. Bring the volume up to 2.5 milliliters using complete RPMI 1640 medium and gently pipette two to three times to mix.Place the opened tube into the magnet and incubate it at room temperature for two and a half minutes. Then pour off the supernatant containing untouched B-cells into a new 15 milliliter conical tube. Use trypan blue and a hemocytometer to count the cells.Re-suspend the purified B-cells in complete RPMI 1640 medium at a final concentration of 0.3 x 10 to the 6th cells per milliliter. Then, to the wells of a 48 well plate, add one milliliter of purified B-cells at a 0.3 x 10 to the 6th cells per milliliter three microgram per milliliter of LPS from E.Coli, five nanogram per milliliter of IL4 and either zero or 500 micromolar HDI. Incubate the cells at 37 degree celsius and 5%carbon dioxide for 60 hours for QRT PCR and high throughput mRNA and miRNA sequencing analysis or 96 hours for flow cytometry analysis.After 96 hours of culture, detach the cells from each well by pipetting them up and down several times. Transfer the cell suspension into a 1.5 milliliter microcentrifuge tube. Then, using a bench top centrifuge, spin down the cells at 350 x G for five minutes.Then, discard the supernatant. Mix a cocktail of HBSS buffer and the following reagents, 1%BSA, 0.5 nanograms per milliliter of conjugated goat anti-mouse IgM antibody, 0.2 nanograms per milliliter of conjugated rat anti-mouse IgG1 monoclonal antibody, 0.2 nanograms per milliliter of conjugated rat anti-mouse B220 monoclonal antibody, 0.2 nanograms per milliliter of PE-Cy7-conjugated rat anti-mouse CD138 monoclonal antibody and two nanograms per milliliter of 7-AAD. Incubate the cells in the dark at room temperature for 30 minutes.Then use one milliliter of HBSS with 1%BSA to wash the cells. Use a bench top centrifuge to spin down the cells at 1, 500 x G for five minutes. After discarding the supernatant, use 300 microliters of HBSS with 1%BSA to re-suspend the cells and transfer the cell suspension to a round bottom polystyrene tube.Cover the tube with foil to avoid light exposure. To carry out QRT PCR analysis of mRNA, include the DNAse 1 treatment step. Then, use a commercial total RNA isolation kit that can recover small RNA to extract RNA from 0.2 to 5 x 10 to the 6th cells, following the manufacturer's instructions.After synthesizing cDNA and performing QRT PCR on the mRNA, carry out RT PCR for microRNA using a cyber green real time PCR master mix by thawing at room temperature, 2x PCR master mix, 250 nanomolar miRNA specific forward primer and a universal reverse primer. After preparing a 25 microliter reaction mix according to the text protocol, add 22.5 microliter aliquots per well to a 96 well PCR plate. Then, add 2.5 microliters of template cDNA to the individual wells.Tightly seal the plate film. Then centrifuge the plate at 1, 000 x G at room temperature for one minute. Place the plate in the real time cycler and start the cycling program found in the text protocol.Using the protocol demonstrated in this video, purified B-cells incubated without PS NIL4 for 96 hours can induce 30 to 40%of CSR to IgG1 and approximately 10%of plasma cell differentiation. After treatment with HDI, the CSR to IgG1 decreased to 10 to 20%while plasma cell differentiation decreased to approximately 2%HDI mediated inhibition of CSR was further confirmed by decreased numbers of post-recombination I mu C gamma 1 and mature VHDJHC gamma 1 transcripts. As measured by QRT PCR, the expression of Aicda which is critical for CSR SHM and Prdm1 and Xbp1, which are important for plasma cell differentiation were shown to be inhibited by VPA.As measured by mRNA sequencing of B-cells stimulated without PS Nil4, only 0.3%of these highly expressed mRNAs were upregulated by HDI more than two fold and only 0.36%of the highly expressed mRNAs, including Aicda, Prdm1 and Xbp1 were reduced by HDI more than 50%The miRNA sequencing analysis of miRNA profiling in B-cells treated with HDI or Nil, showed that HDIs selectively upregulate Aicda and Prdm1 targeting miRNAs. Once mastered, this technique can be done in four and half to five hours. While attempting this procedure, it is important to remember to keep a sterile work environment.After watching this video, you should have a good understanding of how to induce B-cells to undergo class-switching and plasma cell differentiation and to modulate these functions by epigenetic regulators.

genome-wide-analysis-hdac-inhibitor-mediated-modulation-micrornas

Research

JoVE Journal

Immunology and Infection

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Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

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.

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

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

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In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Assays for Validating Histone Acetyltransferase Inhibitors

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Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Learn how to induce B lymphocytes to undergo antibody class-switching and plasma cell differentiation using HDAC inhibitors. This method reveals epigenetic modulation of microRNAs and mRNAs in B cells.

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation (Video) | JoVE Journal

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

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