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 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' 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' 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.
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
2. High-Throughput mRNA-Seq
3. High-Throughput miRNA-Seq
4. Quantitative RT-PCR (qRT-PCR) of mRNAs and miRNAs
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 hallmarks of completed CSR. 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 (Figure 2).
The modulation of mRNA expression by HDI in B cells was highly selective. As measured by mRNA-seq in B cells stimulated with LPS and IL-4, only 0.3% of these highly expressed mRNAs were upregulated by HDI more than two-fold, and only 0.36% of the highly expressed (>20 copies/cell in B cells without HDI treatment) mRNAs, including Aicda, Prdm1, and Xbp1, were reduced by HDI more than 50% (Figure 3).
The downregulation of Aicda, Prdm1, and Xbp1 expression could potentially be mediated by miRNAs, which are negative regulators of gene expression. By using miRNA targeting prediction tools (TargetScan.org, miRNA.org, and miRbase.org), we identified multiple miRNAs that can potentially target Aicda or Prdm1. The miRNA-seq analysis of miRNA profiling in B cells treated with HDI or nil showed that HDIs selectively upregulate Aicda- and Prdm1-targeting miRNAs (Figures 4 – 6).
Figure 1. Surface expression of B-cell marker B220, surface antibody IgG1, and plasma-cell marker CD138 were analyzed by flow cytometry.
B220+ IgG1+ cells were B cells switched to IgG1. B220lowCD138+ cells were plasma cells. The percentage of IgG1-switched B cells and plasma cells from B cells stimulated with LPS and IL-4 in the presence of nil or HDI (VPA, 500 M) for 96 h are indicated as the numbers within the gates. Please click here to view a larger version of this figure.
Figure 2. Expression of hallmarks of completed CSR; mature VHDJH-Cγ1 transcripts and post-recombination Iμ-Cγ1 transcripts; and Aicda, Prdm1, and Xbp1 were analyzed by qRT-PCR, normalized to Cd79b transcripts, and shown in a histogram.
Gene expression in B cells stimulated with LPS and IL-4 in the presence of 500 µM VPA and relevant to the gene expression in B cells with the same stimuli in the presence of nil were depicted as 1. The data are from three independent experiments. p values, paired t-test. Please click here to view a larger version of this figure.
Figure 3. B cells were stimulated with LPS and IL-4 and treated with HDI (VPA, 500 µM) or nil for 60 h. mRNA expression was analyzed by mRNA-seq.
(A) Average mRNA expression levels in three independent experiments (reads per kilobase per million mapped reads, RPKMs) were depicted in scatter plots. Each plot corresponds to one individual mRNA expression level. The two dashed lines are two-fold lines. Scatter plots located above the top dashed line or below the bottom dashed line indicate mRNAs that express more than twice or less than half when induced by VPA and nil, respectively. (B) The bar graphs depict the average change in mRNA expression levels (average RPKMs from three independent experiments) in LPS and IL-4-stimulated B cells treated with HDI, as compared to those in B cells treated with nil. Only the mRNAs at an average RPKM >20 in LPS and IL-4-stimulated B cells treated with nil are included. The mRNA expression in each individual experiment, as shown by a scatter plot (C) or bar graph (D), are depicted in the same way as in (A) and (B). p values, paired t-test. Please click here to view a larger version of this figure.
Figure 4. B cells were stimulated with LPS and IL-4 and treated with HDI (VPA, 500 µM) or nil for 60 h. miRNA expression was analyzed by miRNA-seq.
(A) The change in the average miRNA expression levels in B cells treated with HDI as compared to that in B cells treated with nil were depicted by bar graphs. (B) The change in miRNA expression levels in B cells treated with HDI as compared to that in B cells treated with nil in three independent experiments were depicted by bar graphs. Only the miRNAs at average RPM >0.5 in LPS and IL-4-stimulated B cells treated with nil are included. Please click here to view a larger version of this figure.
Figure 5. HDI increases the expression of Aicda-targeting miRNAs, as shown by miRNA-seq.
(A) Sequence alignment of the miRNAs and their target sites in the 3'UTR of Aicda mRNAs. (B) B cells were cultured with LPS and IL-4 in the presence or absence of HDI (VPA, 500 M) for 60 h. The expression of the miRNAs that were predicted to target Aicda 3'UTR were analyzed by miRNA-seq and depicted as RPM. p values, paired t-test. Please click here to view a larger version of this figure.
Figure 6. HDI increases the expression of Prdm1-targeting miRNAs, as shown by miRNA-seq.
(A) Sequence alignment of the miRNAs and their target sites in the 3' UTR of Prdm1 mRNAs. (B) B cells were cultured with LPS and IL-4 in the presence or absence of HDI (VPA, 500 M) for 60 h. Expression of the miRNAs that were predicted to target Prdm1 3' UTR were analyzed by miRNA-seq and depicted as RPM. p values, paired t-test. Please click here to view a larger version of this figure.
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, such as HDIs.
The epigenetic modulate may impact many different cell functions, such as cell proliferation and viability, which could affect CSR and plasma cell differentiation. Therefore, cell proliferation, viability, and the cell cycles of the B cells, with or without HDI treatment, should be analyzed. One of the challenges for analyzing the modulation of mRNA and miRNA expression by HDI or other epigenetic regulators by qRT-PCR is choosing a suitable housekeeping gene or small RNA. Many common housekeeping genes can be altered to varying degrees by epigenetic modulators. The expression levels of many different housekeeping genes should be measured and used to normalize the specific gene expression by qRT-PCR. This issue can also be addressed by mRNA-seq and miRNA-seq, where the expression of individual mRNAs or miRNAs can be normalized by genome-wide mRNA or miRNA levels.
mRNA-seq can not only define the mRNA expression profile with an appropriate bioinformatics pipeline, but this approach can also define the long noncoding RNA (lncRNA) profile. mRNA-seq typically involves the enrichment of poly(A)+ RNAs by oligo(dT) selection. As a number of lncRNA are known to lack poly(A) tails, the mRNA-seq approach involving oligo (dT) selection cannot determine the complete information of lncRNA expression. In order to achieve complete coverage of both mRNA and lncRNA expression profiles, an RNA-seq approach involving rRNA depletion should be used. In most of our experiments, we use a commercial kit to extract the total RNA, including miRNA, for miRNA-seq, mRNA-seq, and qRT-PCR. Prior to constructing a high-throughput sequencing library, the total RNA quality is validated by running an aliquot on an automated RNA, DNA, and protein sample QC system. It is recommended that RNA samples have an RIN number of 7.0 or higher if they are to be used for small RNA library preparation. Samples with lower RIN numbers have a higher percent of degraded RNA products in the size range of small RNA species (15-30 nucleotides). When the RIN number is below 7.0, the coverage will not be as good. Another option for less-than-ideal RNA samples is to perform an rRNA depletion approach rather than an mRNA isolation approach, but this requires that the samples be at least 10 ng/mL.
The protocols for mRNA-seq used here are optimized for 100 – 1,000 ng of total RNA. For miRNA-seq using less than 100 ng of total RNA, small RNA-seq library preparation should follow a more sensitive small RNA-seq preparation protocol, which takes advantage of the natural structure common to most known microRNA molecules. Most mature miRNAs have a 5'-phosphate and a 3'-hydroxyl group as a result of their biogenesis pathway. Because of this, the 3' and 5' adapters in this kit are directly ligated to miRNAs.
To analyze the impact of HDI on B-cell mRNA and miRNA expression in vivo, mice can be treated with VPA or other HDIs by adding this HDI to the drinking water, and the intraperitoneal injection of these mice with T-dependent antigen NP-CGG or T-independent antigen NP-LPS can be performed. The B cells can be purified from the spleen 10 days after immunization.
The authors have nothing to disclose.
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.
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 |