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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 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.
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....
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
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 tr.......
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,.......
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.
....Name | Company | Catalog Number | Comments |
C57BL/6 mice | Jackson Labs | 664 | |
Corning cellgro RPMI 1640 Medium (Mod.) 1X with L-Glutamine (Size: 6 x 500mL; With L-Glutamine) | Fisher Scientific | MT 10-040-CV | |
FBS | Hyclone | SH300 | |
HyClone Antibiotic Antimycotic Solution 100 mL | Fisher Scientific - Hyclone | SV3007901 | |
β-Mercaptoethanol | Fisher Scientific | 44-420-3250ML | |
Falcon Cell Strainers | Fisher Scientific | 21008-952 | |
Trypan Blue Stain 0.04% | GIBCO/Life Technologies/Inv | 15250 | |
ACK Lysis Buffer | Fisher Scientific | BW10-548E | |
Hausser Scientific Bright-Line Counting Chamber | Fisher Scientific | 02-671-51B | |
EasySep Magnet | Stem Cell Technologies | 18000 | |
Falcon Round-Bottom Polystyrene Tubes with Cap | Fisher Scientific | 14-959-1A | |
EasySep Mouse B cell Isolation Kit | Stem Cell Tech | 19854 | |
BD Needle Only 18 Gauge 1.5 inch SHORT BEVEL 100/box | BD Biosciences | 305199 | |
PE/Cy7 anti-mouse CD138 (Syndecan-1) Antibody | BioLegend | 142513 (25 ug) | |
PE-Cy7 B220 antibody | BioLegend | 103222 | |
7-AAD (1 mg) | Sigma Aldrich | A9400-1MG | |
APC anti-mouse/human CD45R/B220 antibody | Biolegend | 103212 | |
Mouse APC-IgG1 200 µg | Biolegend | 406610 | |
FITC anti-mouse IgM Antibody | Biolegend | 406506 | |
FITC anti-mouse/human CD45R/B220 Antibody | Biolegend | 103206 | |
PE Anti-Human/Mouse CD45R (B220) (RA3-6B2) | Biolegend | 103208 | |
HBSS 1X | Fisher Scientific | MT-21-022-CM | |
Bovine Serum Albumin, Fraction V, Heat Shock Treated | Fisher Scientific | BP1600-100 | |
LPS 25mg (Lipopolysaccharides from Escherichia coli 055:B5) | Sigma Aldrich | L2880-25MG | |
Recombinant mouse IL-4 (carrier-free) | BioLegend | 574302 (size: 10 ug) | |
Valproic acid sodium salt | Sigma Aldrich | P4543 | |
SterilGARD e3 Class II Type A2 Biosafety Cabinet | The Baker Company | SG404 | |
Large-Capacity Reach-In CO2 Incubator | Thermo Scientific | 3950 | |
Isotemp Digital-Control Water Baths: Model 205 | Fisher Scientific | 15-462-5Q | |
5mL Round Bottom Polystyrene Test Tube | Fisher Scientific | 14-959-5 | |
Corning CentriStar 15ml Centrifuge Tubes | Fisher Scientific | 05-538-59A | |
1.7 mL Microtube, clear | Genesee | 22-282 | |
Higher-Speed Easy Reader Plastic Centrifuge Tubes 50ml | Fisher Scientific | 06-443-18 | |
ELMI SkySpin CM-6MT | ELMI | CM-6MT | |
Rotor 6M | ELMI | 6M | |
Rotor 6M.06 | ELMI | 6M.06 | |
Drummond Portable Pipet-Aid XP Pipet Controller | Drummond Scientific | 4-000-101 | |
25 mL serological pipette tips | Fisher Scientific | 89130-900 | |
10 mL serological pipette tips | Fisher Scientific | 89130-898 | |
5 mL serological pipette tips | Fisher Scientific | 898130-896 | |
48-well plates | Fisher Scientific | 07-200-86 | |
Allegra 6 Benchtop Centrifuge, Non-Refrigerated | Beckman Coulter | 366802 | |
GH-3.8A Rotor, Horizontal, ARIES Smart Balance | Beckman Coulter | 366650 | |
Allegra 25R Benchtop Centrifuge, Refrigerated | Beckman Coulter | 369434 | |
TA-15-1.5 Rotor, Fixed Angle | Beckman Coulter | 368298 | |
Fisher Scientific AccuSpin Micro 17 | Fisher Scientific | 13-100-675 | |
Fisher Scientific Analog Vortex Mixer | Fisher Scientific | 02-215-365 | |
miRNeasy Mini Kit (50) | Qiagen | 217004 | |
Direct-zol RNA MiniPrep kit | Zymo Research | R2050 | |
Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology) | Fisher Scientific | BP1145-1 | |
Rnase-Free Dnase set (50) | QIAGEN | 79254 | |
NanoDrop 2000 Spectrophotometers | Thermo Scientific | ND-2000 | |
Superscript III First-strand Synthesis System RT-PCR | Invitrogen | 175013897 | |
iTaq Universal SYBR Green Supermix | Bio-rad | 172-5121 | |
Fisherbrand 96-Well Semi-Skirted PCR Plates, case of 25 | Fisher | 14-230-244 | |
Microseal 'B' Adhesive Seals | Bio-Rad | MSB-1001 | |
MyiQ Optics Module | Bio-Rad | 170-9744 | |
iCycler Chassis | Bio-Rad | 170-8701 | |
Optical Kit | Bio-Rad | 170-9752 | |
BD LSR II Flow Cytometry Analyzer | BD Biosciences | ||
FACSDiva software | BD Biosciences | ||
FlowJo 10 | BD Biosciences | ||
2100 Bioanalyzer | Agilent Technologies | G2943CA | |
S200 Focused-ultrasonicator | Covaris | S200 | |
SPRIworks Fragment Library System I for Illumina | Beckman Coulter | A288267 | |
cBot Cluster Generation Station | illumina | SY-312-2001 | |
HiSeq 2000 Genome Sequencer | Illumina | SY-401-1001 | |
TruSeq RNA Library Prep Kit v2 | Illumina | RS-122-2001 | |
TruSeq Small RNA Library Prep Kit | Illumina | RS-200-0012 | |
NEXTflex Illumina Small RNA Sequencing Kit v3 | Bioo Scientific | 5132-05 | |
2200 TapeStation | Agilent | G2964AA |
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