Expression and methylation data from SLE patient and healthy control blood samples subdivided with respect to ARID3a levels.

Previously published studies revealed that variation in expression of the DNA-binding protein ARID3a in B lymphocytes from patients with systemic lupus erythematosus (SLE) correlated with levels of disease activity ("Disease activity in systemic lupus erythematosus correlates with expression of the transcription factor AT-rich-interactive domain 3A" (J.M. Ward, K. Rose, C. Montgomery, I. Adrianto, J.A. James, J.T. Merrill et al., 2014) [1]). The data presented here compare DNA methylation patterns from SLE peripheral blood mononuclear cells obtained from samples with high numbers of ARID3a expressing B cells (ARID3a(H)) versus SLE samples with normal numbers of ARID3a(+) B cells (ARID3a(N)). The methylation data is available at the gene expression omnibus (GEO) repository, "Gene Expression Omnibus: NCBI gene expression and hybridization array data repository" (R. Edgar, M. Domrachev, A.E. Lash, 2002) [2]. Isolated B cells from SLE ARID3a(H) and ARID3a(N) B samples were also evaluated via qRT-PCR for Type I interferon (IFN) signature and pathway gene expression levels by qRT-PCR. Similarly, healthy control B cells and B cells stimulated to express ARID3a with the TLR agonist, CpG, were also compared via qRT-PCR. Primers designed to detect 6 IFNa subtype mRNAs were tested in 4 IFNa, Epstein-Barr Virus-transformed B cell lines ("Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anemia type I" (S.H. Wickramasinghe, R. Hasan, J. Smythe, 1997) [3]). The data in this article support the publication, "Human effector B lymphocytes express ARID3a and secrete interferon alpha" (J.M. Ward, M.L. Ratliff, M.G. Dozmorov, G. Wiley, J.M. Guthridge, P.M. Gaffney, J.A. James, C.F. Webb, 2016) [4].

Specifications Table

Value of the data

DNA gene methylation data derived from SLE peripheral blood mononuclear cells were subdivided based on levels of ARID3a expression, a transcription factor which correlated with disease activity indices [1], allowing comparison of patient samples with high and low ARID3a levels.

Data for expression of a subset of IFNa associated genes obtained from SLE samples with high or low ARID3a expression, and from healthy control blood cells or those stimulated to express increased levels of ARID3a, allow comparison of effects of high and low ARID3a expression on gene expression.

Data provide validation of primer sets useful for studying Type I interferon signature genes.

Data

One database link, three tables, and one figure are provided in this article. Methyl-seq data from SLE PBMCs segregated based on high or normal numbers of ARID3a+ B cells was deposited in NCBI׳s GEO database under the following accession number GEO: GSE84965 [2]. Table 1, Table 2 show qRT-PCR data obtained via Biomark HD for Type I IFN pathway genes from RNA derived from SLE B cells subdivided based on ARID3a levels [1], and for healthy control B cells with or without CpG induced ARID3a expression [4]. IFN signature genes are in bold. Primers for RT-PCR and qRT-PCR are given in Table 3. Fig. 1 shows the results of RT-PCR of IFNa in four EBV-transformed lymphoblastoid B cell lines [3].

Table 1

Upregulated genes in ARID3aH versus ARID3aN SLE B cells.

Gene	ARID3aH	ARID3aN	P-value
ARID3a	0.6843	0.0692	0.0008
BCL2	4.6000	1.3283	0.0081
BCL2L1	7.6980	0.3608	0.0049
EPSTI1	1958.1900	17.6625	0.0034
HERC5	17.4250	1.9833	0.0043
IFI6	46.0745	3.7483	0.0227
IFI27	1958.1900	17.6625	0.0034
IFI44	23.6242	16.6467	0.3387
IFI44L	130.6440	29.8092	0.0369
IFIT3	33.2118	14.8208	0.0691
IFNA2	49.0570	22.9925	0.2194
IFNAR1	1.0425	1.2433	0.5319
IFNB1	9.3942	1.1575	0.0004
IRF3	2.1863	0.1408	0.0008
IRF5	1.3742	0.3783	0.0035
IRF7	3.5360	0.2510	0.0006
ISIG15	5.6233	0.6150	0.0118
Ly6E	21.4388	1.5683	0.0023
MX1	33.6467	4.3542	0.0009
MYD88	4.1236	2.2740	0.0999
OAS1	11.7725	0.3267	0.0007
OAS2	0.8475	0.2542	0.0847
OAS3	6.0257	0.2313	0.0018
PLSCR1	17.5975	0.8400	0.0526
SIGLEC1	144.2100	55.8713	0.0570
STAT1	2.2225	1.0158	0.0660
TLR 7	5.7500	2.0808	0.0165
TLR9	3.8250	2.8425	0.4706
USP18	17.5975	0.8400	0.0010

Table 2

Upregulated or downregulated genes in CpG-stimulated versus unstimulated healthy control B cells.

Gene	CpG	Unstimulated	P-value
Upregulated
EPST1	3.14125	1.01875	<0.000001
HERC5	6.9225	0.9875	<0.000001
IFI6	1.95	1.00125	0.001894
IFI27	4.9	1.58	0.001146
IFI44	1.805	1.05125	0.003712
IFI44L	2.2325	1.23	0.001263
IFIT3	3.06375	1.19625	0.000121
IFNA2	3.16625	1.07125	0.015070
IFNAR1	2.12	1.0075	<0.000001
IFNB1	3.3125	1.325	0.017893
IRF3	1.94875	1.01875	<0.000001
IRF5	1.01375	1.04125	0.710859
IRF7	1.5325	0.98625	<0.000001
ISG15	0.7825	0.8975	0.385249
Ly6E	1.6825	1.015	0.000261
MX1	2.61125	1.0375	0.000002
MYD88	1.31125	0.96625	0.000083
OAS1	4.3175	0.945	<0.000001
OAS2	1.7975	0.99	0.000954
OAS3	2.1375	1.0725	0.001380
PLSCR1	1.5125	0.99625	0.000332
STAT1	1.0025	1.01375	0.808948
TLR7	5.30625	1.0375	0.000002
TLR9	2.28375	0.99875	0.001518
Downregulated
BCL2L1	0.7475	0.9525	0.009423

IFN signature genes are in bold.

Table 3

Primer sequences.

Gene	Primer sequence (5′ to 3′)	Figure
IFIT1	CTCCTTGGGTTCGTCTATAAATTGAGTCAGCAGCCAGTCTCAG	Fig. 1a in [4]
HPRT1	TTGGTCAGGCAGTATAATCCGGGCATATCCTACAACAAAC	Fig. 1a in [4]
GAPDH	GCCGCATCTTCTTTTGCGTGCCCAATACGACCAAATCCGT	Fig. 1a in [4]
CMYC	ACTCTGAGGAGGAACAAGAATGGAGACGTGGCACCTCTT	Fig. 1e in [4]
ARID3A	AACAAGAAGCTGTGGCGTGATCATGTATTGGGTCCGCAGG	Fig. 1c in [4]
ACTIN	ATCTGGCACCACACCTTCTACAATGAGCTGCGCGTCATACTCCTGCTTGCTGATCCACATCTGC	Fig. 1
IFNA	CCTGGCACAAATGAGGAGAAAGCTGCTGGTAAAGTTCAGTATAG	Fig. 2a in [4]Fig. 1
OAS1	TACCCTGTGTGTGTGTCCAAAGAGGACTGAGGAAGACAACC	Fig. 3a, 4d in [4]Table 1
OAS2	TGGTGAACACCATCTGTGACCCATCGGAGTTGCCTCTTAA
OAS3	AGGACTGGATGGATGTTAGCCACTTGTGGCTTGGGTTTGAC	Table 1
ISG15	CTGAGAGGCAGCGAACTCAGCTCAGGGACACCTGGAA	Table 1
PLSCR1	GTTGTCCCTGCTGCCTTCATGGGTGCCAAGTCTGAATAACA	Table 2
HERC5	TTCAGATCACATGTGGAGATTACCGTTCTGTCCCCAGGCAAAA	Table 1, Table 2
IFI44	GGCTTTGGTGGGCACTAATATGCCATCTTTCCCGTCTCTA
IFIT3	ACTGGCAATTGCGATGTACCGCTCAATGGCCTGCTTCAAA	Table 2
LY6E	TGCTCCGACCAGGACAACTAGGCTGTGGCCAAATGTCAC	Table 1, Table 2
MX1	ATGCTACTGTGGCCCAGAAAGGCGCACCTTCTCCTCATA	Table 1, Table 2
USP18	TGAATGTGGACTTCACCAGGATAGCAGCAGAAGCATCTGGAAA	Table 1
IFI44L	GCAAAAGTGAAGCAAGTTCACAGAACCTCACTGCAATCATCCA	Table 1, Table 2
IFI6	TGCTACCTGCTGCTCTTCATCAGGGCCTTCCAGAACC	Table 1
SIGLEC1	AGGAGGCGTGTTTGTAAGCATGTGGCTGCATCAGGATCAA	Fig. 3a in [4]
IFI27	TTGTGGCTACTCTGCAGTCACCCAGGATGAACTTGGTCAA	Table 1
EPSTI1	GCAAGAGCAAGAAAGAGCCAAACCTTGGAGTCGGTCCAGAAAA	Table 1, Table 2
IRF3	ACCAATGGTGGAGGCAGTACTGGGGCCAACACCATGTTA	Fig. 3b, 4e in [4]
IRF5	AGATCTACGAGGTCTGCTCCAACCTCTCCTGCACCAAAAGAGTA
IRF7	GGCAGAGCCGTACCTGTCAACCGTGCGGCCCTTGTA
TLR7	TCTTCAACCAGACCTCTACATTCCAGCCCCAAGGAGTTTGGAAA	Table 1, Table 2
TLR9	TGCAACTGGCTGTTCCTGAAACAAGGAAAGGCTGGTGACA	Table 2
MYD88	CTGCAGAGCAAGGAATGTGACTGCTGGGGAACTCTTTCTTCA
IFNAR1	AGTGACGCTGTATGTGAGAAAAACGGGAGAGCAAATAATGCA	Fig. 3b in [4]Table 2
STAT1	ATGCTGGCACCAGAACGAAGCTGGCACAATTGGGTTTCAA
IFNA2	AGGATTCAGCGGGAACACAACAATCTCAAACTCTGGTGGTTCAAA	Table 2
IFNB1	ATGAGCAGTCTGCACCTGAAGACTGTACTCCTTGGCCTTCA	Table 2

Fig. 1

EBV-transformed lymphoblastoid B cell lines (LCLs) express IFNa. RT-PCR analysis of IFNa expression in 4 distinct EBV-transformed lymphoblastoid lines was measured in comparison to the positive control cell line, 293T. A no template (NT) negative control is also shown. The housekeeping gene, β-actin, was amplified to demonstrate relative levels of IFNa RNA in each cell line.

Experimental design, materials and methods

Peripheral blood cells and cell lines

Total peripheral blood mononuclear cells (PBMCs) were obtained via Ficoll purification, and were stained for the pan-B cell marker CD20 and intracellular ARID3a prior to analyses by flow cytometry, as previously described [1]. These data allowed subdivision of SLE samples into ARID3a high and ARID3a normal patient samples, such that ARID3aH SLE samples had numbers of ARID3a+ B cells >2 standard deviations above the average numbers of ARID3a+ B cells in healthy controls (>9830 ARID3a+ B cells/ml), versus ARID3aN (<9830 ARID3a+ B cells/ml), as defined previously [1]. B lymphocytes purified by flow cytometric sorting (>97% purity via post-sort analyses) were used immediately for RNA preparation in the case of SLE samples, or in the case of healthy control cells, were grown in complete RPMI media (RPMI 1640, 5×10−5 M β-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 1 mM sodium pyruvate) supplemented with 4% heat inactivated fetal bovine serum (FBS), with or without 5 µg/ml Class CpG oligonucleotide for 24 h, as previously described [4]. Epstein-Barr Virus (EBV)-transformed lymphoblastoid B cell lines (LCLs) were generated from 4 SLE patient samples and maintained in complete RPMI media.

Methyl-seq

To determine if increased expression of ARID3a within SLE patient samples was associated with alterations in DNA methylation, genomic DNA was isolated using standard phenol/Chloroform extraction protocols from total PBMCs obtained from each of two SLE patient samples characterized as ARID3aH and two independent SLE samples characterized as ARID3a low. DNA was fragmented on a Covaris S2 sonicator (Covaris, Woburn, MA) to an average size of ~350 bp in length and methylated DNA was isolated using the MethylMiner Methylated DNA Enrichment Kit (Life Technologies, Carlsbad, CA). Illumina sequencing libraries were prepared from each sample using the Illumina Truseq DNA LT Sample Prep Kit (Illumina, San Diego, CA) by the Genomics Core facility at Oklahoma Medical Research Foundation. Libraries were sequenced on an Illumina Hiseq 2000 instrument with paired-end 100 bp reads. Quality control metrics were assessed with Picard tools v. (https://broadinstitute.github.io/picard/). After sequencing, reads were aligned to the human reference genome hg19 using the aligner BWA-MEM [5] followed by local realignment around problematic indel sequences using the Genome Analysis Tool Kit (GATK) [6]. Genes with statistically significant methylation differences were defined using EpiCenter v. 1-6-1-8 [7]. Methylation differences were tested over promoters of the genes, defined as 2000 bp regions upstream of gene’ transcription start sites. The differentially methylated regions were visualized in the IGV integrative genomics viewer [8]. For visualization in the UCSC Genome Browser BigWig files were created from the final BAM files using a combination of BEDTools [9] and UCSC conversion utilities [10].

Biomark HD assays

Peripheral blood mononuclear cells were isolated from peripheral blood of 6 SLE patients and 2 healthy individuals, and were analyzed for ARID3a expression as described above by flow cytometry. B lymphocytes were enriched from the remaining PBMCs via negative selection using magnetic beads containing other lineage markers, and the remaining cells were stained with CD20 for fluorescence activated cell sorting (FACS) using a FACSAria II (BD Biosciences). Post-sort analyses revealed >98% CD20+ B lymphocytes. RNA was isolated, quantified and assessed for integrity using Agilent Total RNA Pico chips on the 2100 Bioanalyzer (Agilent Technologies, Boblingen, Germany). The DELTAgene assay designer was used for primer design for optimal performance on the Biomark HD system. Primer specificity was determined via melting curve analysis at 400 nM. cDNA preparation (Fluidigm preamp master mix, PM100-5580), amplification (Fluidigm, DELTAgene assay kit), qRT-PCR and analyses were all performed as previously described [11]. Data in Table 1, Table 2 are normalized to the housekeeping gene Hprt1. A list of primers for the genes assessed is given in Table 3.

IFNa analyses of EBV lines

For qRT-PCR, RNA was extracted using Tri-Reagent (MRC, Inc.) and chloroform:isoamyl alcohol 24:1 (Sigma), precipitated in isopropanol, and collected via centrifugation. cDNA was synthesized at 37 °C for 1 h with M-MLV reverse transcriptase (Promega) and random primers (Promega), and amplified for 40 cycles at 60 °C for 30 s, 72 °C for 1 min, and 95 °C for 30 s for IFNa (IFNA2, IFNA5, IFNA6, IFNA8, IFNA14, IFNA16) gene expression. Amplified products were electrophoresed through 2% agarose gel.

Subject area	Immunology
More specific subject area	SLE and ARID3a+B cells
Type of data	Figure, Tables, link
How data was acquired	Electrophoresis and BIOMARK HD
Data format	Raw, analyzed
Experimental factors	FACS-purified SLE and healthy B lymphocytes (+/- CpG-stimulation)
Experimental features	DNA was isolated from ARID3aHand ARID3aNtotal PBMCs; RNA was extracted from LCLs, peripheral blood SLE B cells, and healthy control B cells with or without CpG-stimulation for 24 hours.
Data source location	Oklahoma City, OK; USA
Data accessibility	Data is available within this article and deposited in NCBI׳s Gene Expression Omnibushttp://www.ncbi.nlm.nih.gov/geo/ accessiblevia GEO series accession number: GEO:GSE84965