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Systemic Lupus Erythematosus and DNA Methylation

Systemic Lupus Erythematosus and DNA Methylation. Terrence Shin MCB 5255 Dr. Lynes Mar. 28, 2012. Systemic Lupus Erythematosus (SLE). Chronic autoimmune disorder Affects skin, joints, kidneys, and others Joint pain and swelling Lupus nephritis Cause – Unknown Ages: 10~50

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Systemic Lupus Erythematosus and DNA Methylation

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  1. Systemic Lupus Erythematosus and DNA Methylation Terrence Shin MCB 5255 Dr. Lynes Mar. 28, 2012

  2. Systemic Lupus Erythematosus (SLE) • Chronic autoimmune disorder • Affects skin, joints, kidneys, and others • Joint pain and swelling • Lupus nephritis • Cause – Unknown • Ages: 10~50 • More common in women • African Americans and Asians are affected more often

  3. Common Symptoms of SLE • Chest pain • Fatigue • Fever • Hair loss • Mouth sores • Swollen lymph nodes • Skin/Butterfly rash • Photosensitive

  4. Possible Body Parts Targeted by SLE • Skin • Raynaud’s phenomenon • Brain & nervous system • Lungs • Heart • Arrhythmias • Tachycardia • Bradycardia • Digestive tract

  5. Diagnosis of SLE • CBC • ANA • Chest x-ray • Urinalysis • Renal biopsy

  6. Patients with SLE met at least four of revised criteria of the American College of Rheumatology.

  7. Treatment for SLE • No cure • Control symptoms • Anti-inflammatory medication • Corticosteroids • Protection from sunlight

  8. Ultraviolet B exposure of peripheral blood mononuclear cells of patients with systemic lupus erythematosus inhibits DNA methylation GS Wang, M Zhang, XP Li, H Zhang, W Chen, M Kan and YM Wang Lupus 2009

  9. Background • Sunlight can induce exacerbation of SLE • Ultraviolet B (UVB): 290~320 nm • SLE cells are hypomethylated • Promoters of transcriptionally active gene • DNA methylation • Suppressive effects on gene expression • 5th position of the cytosine ring • Catalyzed by DNMT1

  10. Specific Aims • What are the effect of UVB on DNA methylation in SLE? • What is its significance in the pathogenesis of SLE ?

  11. Materials & Methods • Study participants • Isolation of PBMCs • Irradiation protocol • DNA extraction and HPCE • RNA isolation and RT-PCR • Statistical analysis

  12. Study Participants • 45 patients with SLE • 40 females, 5 males • Mean age: 36 years; range: 18~61 years • Active = SLEDAI ≥ 10 (21 out of 45 patients) • 20 healthy volunteers (control) • Sex- and age-matched • 18 females, 2 males • Mean age: 31 years, range: 22~46 years

  13. Isolation of PBMCs • Total volume of 20 ml of ethylenediaminetetraacetic acid (EDTA)-K2-preserved venous peripheral blood drawn from patients & controls • Ficoll gradient centrifugation • PBMCs • PBMCs cultured in RPMI 1640 medium • Supplemented with 10% heat-inactivated FBS, 2 mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin

  14. Irradiation Protocol • UVB irradiation • Waldman UV109B lights with TL-12 lamps (Waldman Lighting Ltd., Germany) • Emit within the UVB range (290–320 nm) with an emission peak at 311 nm • PBMC were irradiated in PBS with different doses of UVB (0, 50 and 100 mJ/cm2) • PBS removed  RPMI 1640 containing 10% FBS added  cultured (24 h)  DNA & RNA extractions

  15. DNA Extraction and HPCE • Five deoxynucleoside standards purchased from International Laboratory (CA, USA) • dA = 2’-deoxyadenosine • dT = 2’-deoxythymidine • dG = 2’-deoxyguanosine • dC = 2’-deoxycytidine • mdC = 5-methyl-2’-deoxycytidine • All nucleosides were dissolved in ddH2O water

  16. DNA Extraction and HPCE (cont.) • DNA extraction • gDNA extraction kit (QIAmp, DNA mini kit; Qiagen, Hilden, Germany) • RNA digestion • Added 20 μg/μl RNase A (Sigma-Aldrich, St. Louis, MO) • DNA hydrolysis • Heated 18 μl of DNA samples for 2 min in boiling water bath  cooled rapidly in ice • Added 4.5 μl of 10 mM ZnSO4 and 7.5 μl of nuclease P1 (Sigma-Aldrich, St. Louis, MO) • Incubated at 37°C for 16 h • Added 7.5 μl of Tris (0.5 M, pH 8.3), and 4.5 μl of alkaline phosphatase (Sigma-Aldrich, St. Louis, MO) and 50 U/ml in 2.5 M (NH4)2SO4 • Incubated at 37°C for 2 h

  17. DNA Extraction and HPCE (cont.) • CE system (Beckman P/ACETM MDQ) • Uncoated fused-silica capillary (60 cm × 75 μm, effective length 57 cm) • Connected to Millennium data-processing station • Buffer: 48 mM NaHCO3 (pH 9.6) containing 60 mM SDS • Constant voltage (20 kV) and temperature (25°C) • Pressure injected for 5 s • Hydrolysed DNA or 0.1 mM free deoxynucleoside standard was injected into a HPCE analysis system equipped with photodiode array (PDA) detection • Absorbance was monitored at 256 nm • Capillary conditioning • Washed with 1 M NaOH (2 min)  washed with 1 mM NaOH (1 min)  filled with the running buffer (3 min) • Buffers and washing solutions were filtered through 0.45 μm pores • Hydrolyzed samples • Injected hydrostatically (30 s) from 9.8 cm above the cathode • Comparison of migration time & peak area of each sample with the standards • Can calculate DNA methylation levels • Quantification of the relative methylation of each DNA sample • mdC/(dC + mdC) x 100%

  18. RNA Isolation and RT-PCR • Total RNA • Isolated from PBMCs • Trizol reagent (Invitrogen, Carlsbad, CA, USA) • Reverse transcription • Reverse Transcription System (Promega, Madison, WI, USA) • cDNA synthesis • 1.0 μg total RNA—single round reverse transcriptase reaction (TV = 20 μl) containing 0.5 μg oligo (dT) 15 primer, 2.0 μl dNTPs, 2.0 μl reverse transcriptase 10x buffer, 15 μl AMV reverse transcriptase and 0.5 μl recombinant RNasin ribonuclease inhibitor.

  19. RNA Isolation and RT-PCR (cont.) • RT-PCR • ABI Prism 7500 Sequence Detection System • Using SYBR Premix Ex Taq Kit (TaKaRa Bio Inc.) • 10 μl of 2x SYBR green Master Mix, 0.2 μM primers, 5 μl 1:5 dilution of prepared cDNA, 20 μl of water • 40 cycles at 94°C (30 s), 60°C (40 s), and 72°C (60 s) • Primers used: • β-actin (forward & reverse), DNMT1 (forward & reverse) • Purchased from TaKaRa Bio Inc

  20. Statistical Analysis • Mann-Whitney U-test or one-way ANOVA • Equality of means—to compare values • Spearman’s rank correlation • Examine the relationship between two continuous variables

  21. Figure 1: Electrophoretogram of nucleosides separated by HPCE • A. Electrophoretogram—standard nucleosides • B. Electropherogram of hydrolyzed gDNA from SLE patients • Five deoxynucleosides could be distinguished under the given conditions (running buffer of 48 mM NaHCO3, at pH 9.6, containing 60mM SDS, with a separation voltage of 20 kV, injection pressure of 0.7 psi, at 25 °C and pressure injection of 5 s)

  22. Figure 2: Calibration curves • A. dC regression B. mdC regression • Different quantities of dC and mdC were added to a mixture of dA, dT and dG (0.1 mM) to obtain the concentrations of dC, and mdC

  23. Figure 3: DNA methylation in SLE patients and controls • Significant decrease in DNA methylation level in A and S compared to C • No difference between A & S SLE patients A=active; S=stable; C=control

  24. All groups showed decreased DNA methylation levels • Active SLE group showed significant decrease after 50 mJ/cm2 • Stabe SLE group showed significant decrease after 100 mJ/cm2 • Control group showed significant decrease after 100 mJ/cm2

  25. Figure 4: Electrophoretograms of nucleosides from irradiated PBMC

  26. Figure 5: DNMT1 mRNA expression of irradiated PBMC • No significant differences could be found in the expression of DNMT1 before and after UVB radiation

  27. Decrease in DNA methylation levels for all patients after UVB irradiation irrespective of their clinical characteristics • Patients with malar rashes have lower DNA methylation levels than patients without rashes • Patients with leucopenia had significantly lower DNA methylation levels after UVB irradiation than that in patients with normal leucocyte numbers • No obvious differences were observed between patients with or without thrombocytopenia

  28. Discussion & Unknowns • SLE patients have significantly lower levels of methylated DNA compared to controls (without UVB irradiation) • Does down-regulation of DNA methylation play a role in the pathogenesis of SLE? • DNA methylation levels decreased after UVB exposure • Especially for patients with facial rash and leucopenia • Does UVB play a role in pathogenesis of SLE by decreasing DNA methylation?

  29. IL-6 Modulates CD5 Expression in B Cells from Patients with Lupus by Regulating DNA Methylation Soizic Garaud, Christelle Le Dantec, Sandrine Jousse-Joulin, Catherine Hanrotel-Saliou, Alain Saraux, Rizgar A. Mageed, Pierre Youinou and Yves Renaudineau The Journal of Immunology, 2009

  30. Background • Features of autoimmunity in SLE • B cell hyperactivity • Spontaneous lymphocyte proliferation • Production of antibodies to self-antigens • B cell abnormalities in SLE • Excess cytokine production • Autoantigen presentation to T cells • Modulation of the function of other immune cells • SLE is considered as a B cell disease • Therapies target B cells

  31. Background (cont.) • CD5 isoforms • CD5-E1A • CD5-E1B • Demethylated in B cells of SLE patients • CD5-E1B mRNA is transcribed at the expense of CD5-E1A mRNA transcription

  32. Specific Aims • Does the engagement of the BCR with constitutive IL-6 down-regulate the level of membrane CD5, which negatively regulates BCR signaling, in SLE B cells? • Does this signal promote the activation and expansion of autoreactive B cells in SLE patients?

  33. Materials & Methods • B lymphocyte isolation • FACS analysis • Cell culture • mRNA extraction and quantitative RT-PCR • RACE • Methylation-specific PCR • Bisulfite sequencing • Chromatin immunoprecipitation (ChIP) • Computational promoter analysis • Statistics

  34. B Lymphocyte Isolation • PBMCs from the blood of 25 SLE patients and HCs • Centrifugation on Ficoll-Hypaque • Active = SLEDAI ≥ 5

  35. B Lymphocyte Isolation (cont.) • Cells permeabilized with 70% methanol  intracellular staining • FITC-anti-CD19 (clone J4-119) and PE-anti-CD5 (clone BL1a) • Obtained from Beckman-Coulter • Anti-DNMT1 and anti-p27kip1 • Obtained from Abcam • CD5-CD19+ B cells • Sorted on an Epics Elite FACS (Beckman-Coulter) • 98% CD19+ • Daudi human B cell line • Purchased from American Type Culture Collection

  36. FACS Analysis • Number of CD5 molecules per cell • Quantum Simply Cellular kit • Amount of Ab binding to the cells (ABC) at saturating concentrations • FACS analysis • 50μl calibrated microspheres stained with 20 μl of the same anti-CD5 Ab • Mean fluorescence intensity  standard ABC curve  arbitrary ABC value

  37. Cell Culture • B cells (sorted via FACS) • Suspended in RPMI 1640 • Supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 200 U/ml penicillin and 100 μg/ml streptomycin. • B lymphocytes were seeded at 2 x 105 cells per well • Incubated with 1 μg/ml anti-IgM Ab-coated Sepharose beads (BioRad) and 10 U/ml IL-2 • In the presence or absence of 10–40 ng/ml anti-IL-6RAb (R&D Systems), or 100 ng/ml rhIL-6 (Immuno Tools). • Repression of DNMTs • Incubated with 50 μM of the ras signal blocker PD98059 • Detected IL-6 and IFN-ɣ in sera • Detected IL-6 in the supernatant of cultured cells using ELISA kits (Beckman Coulter).

  38. mRNA Extraction and qRT-PCR • Total mRNA • Extracted using the RNAble method (Eurobio) • cDNA synthesis • Reverse transcription • 20 μl volume with Superscript II RNase H-RT (Invitrogen Corporation). • Quantitative RT-PCR • Conducted in 20 μl mixtures • 50 ng template cDNA • 1X Sybr Green PCR Master mix (Applied Biosystems) • 500 nM of each primer (Table II) • All assays included a negative and positive controls • CT method • Comparison of cycle thresholds • 18S as an internal control

  39. mRNA Extraction and qRT-PCR (cont.)

  40. RACE • mRNA 5’ transcript ends • Amplified with SMART-RACE kit (Clontech) • cDNA first strand synthesis • Sense UPM primer and the gene-specific antisense primer CD5 E5 (Table II) • PCR protocol • Initial denaturation at 94°C for 5 min  5 touchdown-PCR cycles of denaturation at 94°C for 30 s  Annealing at 72°C for 3 min  Another 5 cycles at 94°C for 30 s, 70°C for 30 s,72°C for 3 min  decreasing temperature for 35 cycles of 94°C or 30 s, 68°C for 30 s, 72°C for 3 min. • Nested PCR • Sense NUP primer and the gene-specific antisense primer CD5 E3 • Second PCR round: • 40 cycles at 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min with a final extension at 72°C for 10 min.

  41. RACE (cont.) UPM Primer NUP Primer

  42. Methylation-specific PCR • Genomic DNA • Purification – QIAmp 96 DNA blood kit (Qiagen) • Digestion – 20 U of the methylation-sensitive REs (HpaII, HaeII, FauI, HgaI) or the methylation insensitive restriction enzyme (MspI) – 37°C for 3 h • Undigested Genomic DNA • Positive control • PCR primers • Positioned upstream and downstream of E1A and E1B promoter recognition sites • E1A and E1B of CD5, CD19 CD70, Pax5, Syk, and HRES-1 genes (Table II) • PCR protocol • Initial denaturation at 94°C for 5 min  35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 1 min, and primer extension at 72°C for 1 min  final extension at 72°C for 10 min. • Products separated on agarose gel • Visualized with 0.5 g/ml ethidium bromide.

  43. Methylation-specific PCR (cont.)

  44. Bisulfite Sequencing • Methylation status of DNA • Non-methylated cytosines were converted to uridines by bisulfite treatment • EZ-DNA methylation-Gold kit (Zymo Research) • Unmodified DNA (100 ng) • Amplified 40 times at 56°C using specific primers • Sequenced using specific primers • Bisulfite-converted DNA • Amplified by nested PCR using two rounds of 40 cycles each at 56°C with primers specific for methylated cytosines (Table II) • PCR products • Purification – high pure PCR product purification kit (Roche) • Sequencing – Internal primers • BigDye Terminator Cycle Sequencing kit using an automated ABI-310 genetic analyzer (Applied Biosystems) • Electrophoregram • Quantification of T and C peaks • Determination of methylation status = [peak (C)/peak (T) + peak (C)] x 100

  45. Bisulfite Sequencing (cont.)

  46. ChIP • EpiQuik kit (Epigentek Group) • Evaluation of the activation of the CD5-E1B promoter • Sonicated DNA (200–1000 bp) • Transferred into strip wells • Precoated with mouse anti-RNA polymerase II, or with a nonspecific mouse IgG, used as a negative control • Incubated for 90 min at room temperature, then washed • Precipitated DNA-protein complexes • Treated with 250 μg/ml proteinase K in the DNA release buffer for 15 min • Left in the same buffer for 90 min at 65°C • Collection of DNA samples • P-spin columns, washed with ethanol, and eluted • PCR • Template – purified DNA • GAPDH & CD5-E1B specific primers (Table II) • 40 cycles at 56°C • Products were separated on agarose gel • Visualized with 0.5 g/ml ethidium bromide.

  47. ChIP (cont.)

  48. Computational Promoter Analysis • Identification of putative transcription factor binding sites • Alibaba (v2.1) • TESS (transcription element search system) • Genomatix

  49. Statistical Analysis • Results as arithmetic means with SD • Mann-Whitney U test • Unpaired data • Wilcoxon test • Paired data • Spearman’s rank correlation

  50. Figure 1: CD5 expression in B cells Scattergrams C. FACS profile of one SLE patient and one HC: cell surface expression of CD5 A. Percentage of CD5-expressing B cells B. Anti-CD5 Ab bound to the cell membrane

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