cocktail for spin infection at 1,290 g for 90 minutes. A second round of spin infection was performed 24 hours following the first one. At least 56105 cells were injected intravenously into lethally irradiated (9 Gy) recipient C57BL/6 mice. Antibiotic containing drinking water was provided for recipient mice for two weeks post transplantation. Cell cultures HEK293 cells [53] were maintained in DMEM with 10% FBS. Murine pro-B-cell line 38B9 and pre-B-cell line 3-1 [54] were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2-mercaptoethanol. Murine plasmacytoma cell line S194 (American Type Culture Collection) was cultured in DMEM with 10% horse serum. Myc3 B lymphoma cells [55] were cultured on a monolayer of gamma-irradiated S17 feeder cells [56] as described previously [55,57]. Murine myeloid cell line 32D (American Type Culture Collection) was cultured in IMDM with 10% FBS, 10% WEHI-3B supernatant and antibiotics. Human pre-B ALL cell lines 697 [58] and JM-1 (American Type Culture Collection) were cultured in C10 lymphocyte media as previous described [59]. Primary B cell cultures Bone marrow cells from C57BL/6 mice were isolated, plated onto OP9 feeder cells and were cultured in IMDM with 10% FBS, 10 ng/mL IL-7, 50 micromolar 2-mercaptoethanol, 16 L- glutamine, 16 non-essential amino acids and 16 penicillin/ streptomycin. Ten days later, 90% of bone marrow-derived cells were B220+IgM2CD43+ by flow cytometry. Antibodies and flow cytometry Antibodies for FACS analyses were obtained from BD Biosciences-Pharmingen or eBioscience and are as follows: CD19 (1D3), c-kit (2B8), Sca-1/Ly-6A/E (E13-161.7), B220 (RA3-B2), Ter119, CD11b, Gr-1/Ly6G, CD16/32, CD34, CD43, AA4.1, sIgMb, CD21, CD 23, CD4, CD8 alpha, TCR beta, NK1.1, and CD25. Acquisition was performed on FACS Calibur FACSCanto or LSRII (BD). Sorting was performed with a FACSAriaII (BD). Data were analyzed with BD FACStation or FlowJo software. Western blots Total cell extracts were prepared in 26 SDS sample buffer (Biorad). Sodium dodecyl sulfate-polyacrylamide gel electropho- resis, western blotting, and peroxidase-based chemiluminescence detection were performed according to standard laboratory protocols. Antibodies were purchased from Santa Cruz (YY1, H414), BD Biosciences (Bcl-xl, 2H12), or Cell Signaling (NFkB2, 4882; cleaved caspase-3, Asp175). Bands were quantified by using ImageJ software. PCR Detection of Ig Rearrangement Genomic DNA from sorted GFP+ splenic B cells from MigR1 or YY1 reconstituted mice were made by DNeasy Blood & Tissue kit (QIAGEN). For heavy chain rearrangements, DNA samples were amplified by PCR reactions with primers described previously [60]. PCR products were separated on 1.5% agarose gels and visualized by Southern blot with an oligo probe (5-AGC CTT CAG GAC CAA GAT TCT CTG CAA ACG-3) detecting the region upstream of JH3. Quantitative real-time PCR RNA was made by the Trizol procedure according to the manufacturer’s specifications (Invitrogen) and subjected to the YY1 Overexpression Negatively Impacts B Cells reverse transcriptase-polymerase chain reaction (RT-PCR) proce- dure. Murine primers for YY1 were: sense CCCACGGTCCCA- GAGTCCA and anti sense TGTGCGCAAATTGAAGTC- CAGT. Other primers for RT-PCR are listed in Table S1. Hprt was used as an internal control or normalization. Hprt primers were: sense CTCCTCAGACCGCTTTTTCC and antisense TA- ACCTCCTTCATCATCGCTAATC. All amplifications crossed intron-exon boundaries to exclude genomic DNA amplification. ChIP Chromatin immunoprecipitation (ChIP) was carried out as previously described [61]. For all ChIP experiments, quantitative PCR analyses with primers shown in Table S2 were performed by real-time PCR using a Roche Lightcycler 1.5. Relative increases were calculated on the basis of pre-immune ChIP sample quantitative PCR values. Relative enrichments for each region are presented as the percentage of input. BrdU assay BrdU was directly added into the culture medium to obtain a final concentration of 50 mM for 45 minutes. Cells were then fixed, permeabilized, and stained with APC conjugated anti-BrDU antibody following the manufacture’s instructions (BrdU flow kit, BD Pharmingen). 7-AAD was added cells for FACS analyses. Microarray analyses Murine pro-B 38B9 cells were retrovirally infected with MigR1 vector or MigR1-FlagYY1 such that nearly 100% of the cells were transduced. 48 hours post infection, RNA samples were prepared using Trizol reagent (Invitrogen), and cDNA was prepared using the Superscript II kit (Invitrogen). The cDNAs were subjected to hybridization with a DNA-Chip mouse genome 430A array (Affymetrix) using the University of Pennsylvania Microarray Facility’s standard protocol (http://www.med.upenn.edu/microarr/ DataAnalysis/Affymetrix/methods.htm). Data analyses were per- formed with Affymetrix Microarray Suite 5.0. The work complies with MIAME guidelines and has been deposited with ArrayExpress (accession number E-MTAB-679). Supporting Information Figure S1 GFP levels correlate with YY1 expression, and exogenous YY1 is well expressed in chimeric mice. (A) 38B9 cells were tranduced with MigR1-FlagYY1 and four days later cells were sorted into low, middle, and high GFP-expressing populations. Cell lysates were evaluated by western blot with anti- YY1 antibody, and blots showed that YY1 expression correlates with the level of GFP expression. (B) 32D myeloid and 38B9 pro-B cells were transduced with MigR1-FlagYY1 and sorted into GFP low and GFP high fractions of identical intensity between the two cells types. Equivalent amounts of cell lysates from untransduced (endogenous) and GFP sorted samples were evaluated by western blot on the same gel with anti-YY1 antibody (414; Santa Cruz Biotechnology) or anti-actin antibody and signals were quantitated with Image J software. The left panel shows the western blot data from the same gel probed at the same time with the same antibodies. The right panel shows Image J quantitation. Endogenous YY1 levels are higher in myeloid cells and increase to slightly higher levels after MigRI-FlagYY1 transduction compared to 38B9 pro-B cells. (C) Exogenous YY1 is expressed at similar protein levels as endogenous YY1 in B cells. GFP+ lymphocytes were sorted from the blood of MigR1-FlagYY1 reconstituted mice 14 weeks post reconstitution and crude cell lysates were made. Western blot was performed for detection of PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e30656