Supplemental Materials and Methods

 

Cell Culture and Retroviral Transduction

Human B cell lines where grown in RPMI-1640 with 10% fetal bovine serum (FBS, Hyclone) and penicillin/streptomycin.  Viral transduction and selection was performed as described (Shaffer et al., 2000).  For induction of the Blimp-1-ERD fusion protein cells were treated with 1uM CdSO4 and 1uM 3-OH tamoxifen (Sigma).   Most infection were done as described in Figure 1.  One of the two BJAB infections was done as follows: cells were infected with retrovirus in the presence of 5 ug/ml of polybrene and, after 2 days of infection, cells were selected with puromycin (1 ug/ml) for 3 days. Surviving cells were then isolated according to the manufacturer’s protocol for Histopaque-1077 purification (Sigma).

Primary splenocytes were cultured in RPMI medium supplemented with 10% heat inactivated fetal bovine serum (FBS, from Gemini Bio-Product, Inc.), 20 mg/ml Gentamicin (Gemini) and 50 mM b-mercaptoethanol and B cells were purified as described (Piskurich et al., 2000).  Cells (106/ml) were stimulated with LPS (10 mg/ml) alone or together with anti-F (ab)'2 (5 mg/ml) (Southern Biotechnology) or IL-4 (5mg/ml) (R&D Systems) for overnight before retrovirus infection.  Phoenix cells (Dr. G. Nolan, Stanford University) were cultured in DMEM medium supplemented with 10% FBS and 20 mg/ml Gentamicin.  Retroviruses were generated as described (Piskurich et al., 2000).  Primary splenic B cells (106) were infected at an m.o.i. of 0.2 to 2 in the presence of 5 ug/ml of polybrene. Three days post-infection, cells were sorted for expression of YFP. BJAB cells were infected with retrovirus in the presence of 5 ug/ml of polybrene and, after 2 days of infection, cells were selected with puromycin (1 ug/ml) for 3 days. Survival cells were then isolated according to the manufactory’s suggested protocol for Hitopaque-1077 purification (Sigma

  Plasmids

VxyPRDIBF1puro or gfp (Blimp-1) was constructed by ligation of its cDNAs into the EcoRI site of Vxy-puro (Shaffer et al., 2000) as follows:  Each construct was FLAG-tagged by inserting a FLAG-encoding oligo in-frame into the BamHI site immediately 5’ of the Blimp-1 stop codon.  (top oligo:5’-GGATCCTGACTACAAGGACGATGACGAC AAGTAAATCGATGTCGACA-3’; bottom oligo:5’-GATCTGTCGACATCGA TTTACTTGTCTGTCATC GTCCTTGTAGTCAG-3’).  The 5’ end of each construct was also reconstructed to contain a Kozac consensus sequence.  For Blimp-1, an 800bp fragment was amplified using a 5’primer: 5’SalI site- NheI site- Kozac sequence- ATG (5’-ACGCGTCGACGCTAGCCCCACCATGAAAATGGACATGGAGGATGC-3’) and a 3’primer: HindIII (5’-CCCAAGCTTTCAAAAGTCTTCTGGCAG-3’), and this fragment was used to replace the corresponding part of the 5’Blimp-1 coding region. To make the ERD-fusion version of Blimp-1 (Blimp-1-ERD), a PCR product encoding the ERD (Shaffer et al., 2000) was amplified (5’primer: (5’-CGCGGATCCTTCTGCTGGAGACATGAGAGCTGCC-3’)  and 3’ primer: (5’-CGCGGATCCACTGTGGCAGGGAAACCCTCTGCCTCCCCCGTG-3’).  This fragment was then cloned into the BAMHI site at the 3’end of Blimp-1. Modified Blimp-1 was removed from pBSK using SalI and cloned into the XhoI site of the IRES-containing retroviral construct Vxy (with either puro or gfp).  Inducible Blimp-1-ERD was similarly cut from pBSK and cloned downstream of the heavy metal-inducible metallothinein promoter of pMEP4 (Invitrogen).  Cells were transformed by electroporation and selected with empirically determined concentrations of hygromycin (GibcoBRL).  Transformants for all constructs were screen for orientation by PCR, sequence verified, and protein expression was confirmed by Western blotting or immunofluoresence (Shaffer et al., 2000) for the FLAG epitope (M2, Sigma, data not shown). 

            pGC-Blimp-1-YFP was cloned as described in (Piskurich et al., 2000).  pGC-TBlimp-YFP and pGC-Pax5-YFP were cloned as described in (Lin, K.-I, et al, 2002)

Flow Cytometry

BJAB, RAJI, and WI-L2 human B cell lines were acutely infected with control (puromycin resistance (puro) or green fluorescent protein (gfp)) viruses or viruses expressing Blimp-1 (with puro or gfp).  For puro constructs, cells were infected and after 24 hrs. selected with puromycin for an additional 48hrs before staining.  For gfp constructs, after 24 hours cells were counterstained with PE-conjugated antibodies against CD19, CD20, CD22, CD27, CD37, CD45, CD53, CD11A, CD83 (BD Pharmingen), MHC II unconjugated + antimouse-PE (BD Pharmingen), and CXCR5-PE (R&D Systems) as described (REF).  For analysis, gfp+ cells were gated and the expression of each surface marker in the PE channel was assessed using Cellquest software.

For staining surface of IgG1 on retroviral transduced splenic B cells treated with LPS+IL4, after 3 days of virus transduction, cells were collected and stained with biotin-labeled IgG1 (Southern Biotechnology) + Streptavidin-Tricolor (CALTAG). The levels of surface IgG1 were determined for YFP+ gated cells.

 

RT-PCR primers

Human Cell Line RT-PCR primers

GAPDH: 5’- GGGCGCCTGGTCACCAGGGCTG-3’

and 5’-GAPDH: GGGGCCATCCACAGTCTTCTG-3’

BCL-6: 5’-TTAGTTCTCAGAA TTCCAGAGGC –3’

and 5’-ACAACATGCTCCATCTGCAGG-3’

c-myc: 5’-AACCAGAGTTTCATCTGCGACCCG-3’

and 5’-TTGTGCTGATGTGTGGAGACGTGG-‘3

syk: 5’-TGCCTCCTCAAGAAGCCCTTCAAC-3’

and 5’-GATTATTCCACCCGCTGACCAAGTC

ICSBP: 5’-TTCTGTGGACGATTACATGGGGATG-3’

and 5’-ACCTCATCAACGCTCCAGCTTGTTG-3’

CD22: 5’-GGTCGGAAGAAGTGTTCCTGCAAG-3’

and 5’-GGTGGCTGCTTGAGAAGTCACATTG-3’

JAW1: 5’-GGATGTATGCCAAAGAGCACGCTG-3’

and 5’-TTGTTGCCCAGGAAGCTATTGTGTC-3’

napsin-2: 5’-ACGCCTCCACAAAACTTCACTGTTG-3’

and 5’-ATGATGTACTCCCCAGCCAGCAAG.

btk: 5’-CAGGCTGAGCAACTGCTAA-3’

and 5’-GGAGCCTTCTTTGATCATC-3’

BLNK: 5’- GCAAGACACTTCCCAGTAA-3’

            and 5’-CTTGTCTGTGACTTGACCC-3’

EBF: 5’-TCCCGGCCCTTGCTAACAC-3’

and 5’-CTTGCAGGCTGTTCCCGTT-3’

AID: 5’-TTCAAAAATGTCCGCTGGG-3’

and 5’- AGCCCTTCCCAGGCTTTGA-3’

Blimp-1: 5’-TCGGGTCGTTTACCCCATC-3’

            and 5’-CACAGCGCTCAGGCCATTA-3’

XBP-1: 5’-CGCTGAGGAGGAAACTGAA-3’

            and 5’-GGGAGGCTGGTAAGGAACT-3’

 

Taqman RT-PCR

 

            All oligos are from Synthegen (Houston, TX).  Primers were used at 300nm, probes at 25 uM, on serial 4- to 5-fold dilutions in triplicate of each RNA (range=200ng-1ng)

 

            hXBP-1 (specific for unprocessed mRNA) primers:

                        Forward-5’-AGCACTCAGACTACGTGCACCTC

                        Reverse-5’-CAGAATGCCCAACAGGATATC

Probe: (FAM/TAMRA) 5’-AGTTGTCACCCCTCCAGAACATCTCCCCAT

 

            hBeta-2 microglobulin control primers:

                        Forward-5’-TGACTTTGTCACAGCCCAAGATA

                        Reverse-5’-AATCCAAATGCGGCATCTTC

            Probe: (FAM/TAMRA) 5’-TGATGCTGCTTACAATGTCTCGATCCCA

 

            Cycles:  48C, 30mins.,  95C 10mins., then 40 cycles of 95C 15 sec. to 60C 1 min.

 

           

 

Primary Mouse Splenocyte RT-PCR primers

 GAPDH: 5'-TTAGCACCCCTGGCC AAGG-3'

and 5'-CTTACTCCTTGGAGGCCATG-3

 PAX5: 5'-GCGTGTTTGAGAGACAGCACT-3'

and 5'-AAGAATACTGAGGGTGGCTGT-3'

J chain: 5'-CTAGGATCATCCCTTCCAC-3'

and 5'TGATACCTAAGTGGGACCA-3'

CD19: 5'-ACAGGACTGGAAGAAGAAG-3'

and 5'-ACTGAATTGAGTGGAGCTG-3'

CIITA: 5'-CCAGGAAAGGGAGCTGGCCACT-3'

and 5'-AGAAGCTGGGCACCTCAAAGAT -3'

c-myc: 5'-GGGCCAGCCCTGAGCCCCTAGTGC-3'

and 5'-ATGGAGATGAGCCCGACTCCGACC-3'

Blimp-1: 5'-GCCAACCAGGAACTTCTTGTGT-3'

and AGGATAAACCACCCGAGGGT-3'

EBF: 5’- TCCCAGCCCTTGCTAACAC-3’

            and 5’- CTTGCAGGCTGTTCCCGTT-3’

Oct-2: 5’- TGGCCATGGGCAAGCTCTA-3’

            and 5’- TCTGAGGTAGGCTTCTGGT-3’

BCL-6:  5’- cacactcgaattcactctg-3’

and 5’- tattgcaccttggtgttgg-3’

Spi-B: 5’- CCACTTTGAGACTCAGGTG-3’

and 5’-ATAGGTGGAGGGGTTAGGA-3’

ICSBP: 5’-TCCGAGAGCTGCAGCAATT-3’

and 5’-CTTAGACGGTGATCTGTTG-3’

Id3: 5’- TGTGCTGCCTGTCGGAACG-3’

and 5’-ACAAGTTCCGGAGTGAGCT-3’

STAT6: 5’- CACTATAAGCCCGAACAGAT-3’

and 5’-CTACCATAGTCACATCTGA-3’

CD79A: 5’-TCATACGCCTGTTTGGGTC-3’

and 5’-CTTCTCATTTTGCCACCGT-3’.

CD22: 5’-GTCCTGTGAGAGTGATGCCAA-3’

and 5’- GATGGCATAACTAACGGTGTC-3’ 

syk: 5’- ggcagctagtggaacatta-3’

and 5’- ttcacagtcccgaagttac-3’

BLNK: 5’- GCAAGACACTTCCCAGTAA-3’

and 5’-CTTGTCTGTGACTTGACCC-3’

btk: 5’-TAGTGACCTGGTACAGAAA-3’

and 5’-CCACTCATACATCTCTATG-3’

JAW1: 5’-AAGCACTGCTGCAGAACGA-3’

and 5’-TCTCAGAAAGGCTGGGCTT-3’

CD20:  5’-CTGGAGCTTATTCAAACTTCC-3’

and 5’- CTTTGGTTGGGAAGATACTCC-3’.

CD37: 5’- AAAGCGAACGAGTCTGAGGAG-3’

and 5’-ACACAGGAATATTGAGAGCGT-3’


CXCR5: 5’-GCAAAACTGTGATCGCTCT-3’

and 5’-GTCACTAAAATGGCCACCC-3’

AID: 5’-TTCAAAAATGTCCGCTGGG-3’

and 5’- AGCCCTTCCCAGGCTTTGA-3’

A1: 5’-caaatctggctggctgacttttc-3’

and 5’-caagtgctgataaccattctcgtc-3’.

Ku70: 5’-GATGCATCTGAAGAAGCCA-3’

and 5’-AGCACAATCTGACGTGTCC-3’

Ku86: 5’- ATCCTGTTGAAAACTTCCG-3’

and 5’-CTTTGGGGGCCAGAAACTT-3’

DNA-PK: 5’- TTGGAGACAGACACCTGAA-3’

and 5’-TGAATCCATGATCCTCCTT-3’)

germline g1 transcript: 5’-GGCCCTTCCAGATCTTTGAG-3’

and 5’-GGATCCAGAGTTCCAGGTCAG-3’

postswitch g1 transcript: 5’-CTCTGGCCCTGCTTATTGTTG-3’

and 5’-GGATCCAGAGTTCCAGGTCAG-3’.

 

 

Reactions were annealed at 55°C and amplified for 35 cycles (except GAPDH, and c-Myc for 30 cycles and STAT6, AID and A1 for 40 cycles). CIITA and CD22 were annealed at 62 °C, 35 cycles.

 

 

ChIP assay.  10 ΄106 WI-L2 cells (control construct (pMEP4), Blimp-1-ERD-expressing, or BCL-6ERD-expressing (Shaffer, et al. 2000)) were harvested after inducing expression of FLAG-tagged proteins for 20 hrs with cadmium and tamoxifen as described above. Briefly, proteins were crosslinked to genomic DNA by treatment with formaldehyde on ice for 20 mins., followed by neutralization, washing, and sonic shearing of the chromatin.  Aliquots of chromatin were set aside as controls for equivalence of starting material, and the remainder was immunoprecipitated using an anti-FLAG antibody (M2, Sigma).  Captured chromatin was extensively washed, digested with proteinase, and precipitated prior to PCR analysis. (Lin et al, 2002 and Yu et al. 2000). PCR primers were: CIITA: 5’-GGTTCCATTGTGATCATCA-3’ and 5’-AAACTCTCCCTGCAAGGTG-3’, Id3: 5’-GGCTCAAGCTTTCTTCTTTTCCCCTGTTGC-3’ and 5’-CCAATTTGCTGTTCGTCTGACCTCCAG-3’, SpiB: 5’-GTGCGTGAATGTCCCTTTG-3’ and 5’-AGGCAAATGTCCCCCTCCT-3’, CSF-1: 5’-CTCTTCCTCCTGATAGCTCCATGA-3’ and 5’-CACTATGTTAGCCAGGATGGTCTC-3’ and were designed to amplify regions of 230-280 bp. All the PCR reactions were annealed at 55°C and amplified for 30 cycles. PCR products were transferred to nylon membranes and hybridized with either g-P32 labeled probes that recognize an internal sequences or a-P32 labeled random primed probe from the PCR-amplified genomic template.

 

 

Electromobility Shift Assay (EMSA). EMSA for Id3 and Spi-B Blimp-1 binding was performed as described (Lin et al, 1997). The sequence of double-stranded oligonucleotides used for probe and competitors are:

c-myc: CGCGTACAGAAAGGGAAAGGACTAGCGCG

CIITA: ACAGTAAGGAAGTGAAATTAATTT

hId3: GCGCTGAGATTGCAGAAGGAGGAGGGAAAGGGGGTTTGAG

mId3: GCGCTGAGATTGCAGAAGAAGGAGGGAAAGGGAGGTCTAA

hSpiB:  GGTGGGAGGGGCAGGGAAAGTGAG

mSpiB: CGCTGGGGGAGGCAGGGAAAGTAGGG

non-specific: AGGAAGCAGGTCATGTGGCAAGG