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Zinc-finger transcriptional repressor protein BCL-6, associated with the pathogenesis of non-Hodgkins B-cell lymphoma, is exclusively found in and necessary for the formation of germinal centers. A recent report suggests that BCL-6 plays an important role in the down-regulation of the expression of several cytokines including MCP-1, MCP-3, and MRP-1 in Macrophages. MCP-1 expression is induced by TNF, along with PDGF, IFN-µ, stress factors, and viral infection. MCP-1 is important in the pathogenesis of atherosclerosis, HIV replication, glomerular nephritis, and allergic chronic inflammatory diseases. We investigated the potential repression effect of BCL-6 on MCP-1 induction by TNF using transient transfection CAT reporter assays, Northern blot analysis, and Real-time PCR. We also used a mutated MCP-1 promoter containing a candidate binding site for BCL-6 at -1550 to determine if repression is dependent on DNA binding. Results demonstrate a slight repression effect of BCL-6 on MCP-1 induction. The creation of retroviral infection vectors should allow for the further analysis required to confirm this phenomenon.
Deregulation of B-Cell Lymphoma 6 (BCL-6), a 95 kD nuclear phosphoprotein, is among the most common rearrangement in non-Hodgkin's B-cell lymphoma accounting for 30% to 40% of new cases and up to 80% of mortalities. This is often due to a translocation of 3q27 in the regulatory region of the gene. The normal role of BCL-6 is not completely understood; except that it is necessary for the formation of germinal centers and that it has an anti-apoptosis effect. Sequence analysis has indicated six zinc finger domains associated with DNA binding activity near the C-terminal. A POZ domain associated with homo- and hetero- dimerization function has been identified near the amino terminal. Recent studies have indicated the effect of BCL-6 on a wide range of genes involved in B cell activation and terminal differentiation, inflammation, and cell cycle regulation. Gene expression modulated by BCL-6 (usually through repression) include CD69, CD44, CXCR4, EBI2, cyclin D2, p27 kip1, Id2, STAT1, ISGF3, 9-27 (Leu 13), 1-8, and blimp 1. BCL-6 also affects the expression of certain cytokines, including the repression of monocyte chemoattractant protein 1 (MCP-1). MCP-1 is a proinflamatory, C-C chemokine that recruits macrophages, T cells, and basophils to areas of infection and disease. It is associated with a variety of diseases including atherosclerosis, HIV replication, glomerular nephritis, and allergic and chronic inflammatory diseases. It is induced by TNF, platelet-derived growth factor, IFN-µ, stress factors, and viral infection. Previously identified repressors include retinoic acid, gluccocorticoids, and estrogen. In vivo genomic footprinting and mutational analysis upstream of the murine MCP-1 has identified a proximal and a distal region separated by about 2.4 kb on the promoter. The proximal region contains three sites that become occupied upon TNF induction of MCP-1: µB-3, site B, and a GC box. The MCP-1 distal regulatory region contains four elements: site A, µB-1, µB-2, and the HS site. To investigate if the induction of MCP-1 by TNF can be repressed by BLC-6, constructs expressing BCL-6, p65, and a CAT reporter construct containing the MCP-1 promoter were transiently transfected into an NIH 3T3 cell line. This line does not express BCL-6 naturally allowing the amount of BCL-6 expression to be controlled through transfection. The samples collected from these transfections were analyzed by Northern Blot analysis, Real Time Q-PCR, and CAT assay. Our results indicate that there may be a repression of TNF-mediated MCP-1 induction following transfection of large amounts of BCL-6. However it does not appear that this repression is dependent upon the potential BCL-6 binding site at –150 of the MCP-1 promoter. Our results also indicate that the repression effect of BCL-6 may be manifested clearer by stimulating with p65 instead of TNF. Further analysis using BCL-6 retroviral infection vectors is suggested.
Plasmid Construction
The pBCL-6 plasmid was created by reverse transcription of mouse spleen total RNA and PCR amplification of the mouse BCL-6 gene. The gene was then ligated into pEcoHis6 through BamHI and HindIII sites. To create the pMZ-2 plasmid, the BCL-6 gene was excised from the pBCL-6 plasmid with NotI and HindIII, filled in with Klenow enzyme, and ligated into pTJ66, which has been linearized with Xho1 and dephosphorilated with calf intestine phosphatase.
Cell Lines and Transient Transfection
NIH 3T3 cells were grown in DME medium (10% BCS) to 50% confluency, washed, trypsinized, and resuspended in RPMI. For CAT reporter assays each transfection used 10µg of reporter plasmid, 1µg of alkaline phosphatase (for normalization), indicated amount of pBCL-6 or vector, and pUC-18 to bring the total to 61µg of DNA. For RNA assays the reporter plasmid was omitted and the total DNA was 51µg. 5µg of p65 DNA was added when p65 was used to induce expression. The cells were electroporated at 0.3 V, 960µFD and grown in a 1:1 mixture of conditioned media and fresh media. For CAT assay of TNF-induced transfection, the cells were allowed to grow for 24 hours then 500 U/ml of TNF was added. The cells were harvested after an additional 12 hours of growth. For CAT assay of p65-induced expression, cells were harvested 48 hours post transfection. For RNA isolation, cells were harvested after 4 hours of treatment with TNF or 24-48 hours post-transfection if induced with p65. CAT Assay The reporter chloramphenicol acetyltransferase (CAT) was assayed using a CAT ELISA kit (Roche). The 96 well plate was read using a Universal Microplate Reader (Biotek instruments). Northern Blot RNA was collected using the RNeasy RNA extraction kit (Qiagen). The RNA was run on a 1.5% denaturing agarose gel at 60 V for 2 hours. The RNA was transferred overnight in 20X SSC onto a Bio-Rad Zeta Probe Membrane followed by hybridization with 32P labeled MCP-1 specific probe. The membrane was exposed to a phospho-plate overnight or autoradiographic film for three days. After exposure the membrane was stripped and reprobed with GAPDH specific probe. 32P labeled probe was prepared using the Random Primed DNA Labeling Kit (Roche).
Real Time Polymerase Chain Reaction
RNA was extracted as for Northern blots. Each sample was reverse transcribed using oligo d(T)16. PCR was performed in the BioRad iCycler with Sybr Green Dye added into the PCR reaction. The threshold cycle method was used to quantitate. Western Blot Samples were run on 10% SDS PAGE, transferred to PVDF Membrane. The membrane was blotted with rabbit polyclonal anti-BCL-6 antibody (1:200, Santa Cruz) then anti-rabbit conjugate horse radish peroxidase (1:3000, Sigma) Retroviral Infection The protocol was followed as described by T.J. Murphy. Briefly, pMZ-2 or pTJ-66 was transfected into Phoenix cells which express viral structural genes (gag, pol, pro, and enf) then grown at 32¼C, 5% CO2. The virus was collected from the conditioned media at 12 hour time points, filtered, and used to infect NIH 3T3 cells. Flow Cytometry Cells were trypsinized and resuspended in PBS containing 3% FBS and 0.1% NaN3. Samples of 10,000 cells were assayed for GFP florescence using a FACSvantage cell sorter.
BCL-6 seems to repress the transcription of MCP-1 induced by TNF Binding to -150 site on MCP-1 promoter is not essential for repression Retroviral infection with MZ-2 does result in expression of GFP and BCL-6
This material is based upon work supported by the Howard Huges Medical Institute under Grant No. 52003071. This research would not have been possible without the invaluable aid and encouragement of all of the members of Jeremy Boss’ lab. A special thanks to Dr. Shantha N. Kumar for assistance with flow cytometry data. Another special thanks to Aaron M. Robida and T.J. Murphy for retroviral materials, protocols, and equipment.
Zuckerman utilized transient transfection to examine the effect of BCL-6 on TNF-induced MCP-1 production. Northern blot analysis examined the RNA levels for MCP-1 induction and demonstrated slight repression. When p65 was used to induce MCP-1 expression instead of TNF the repression effect was easier to see due to lower background. CAT assay which used the MCP-1 promoter attached to a CAT reporter also demonstrated some repression. Mutation of the proposed BCL-6 binding site at the –150 location of the MCP-1 reporter did not effect repression, suggesting that binding at this site is not essential. In order to increase transfection efficiency a retroviral vector was sub cloned by the addition of the BCL-6 gene to the pTJ66 vector. The resulting vector demonstrated successful infection in 53% of the cells as demonstrated by GFP produced by infected cells. Western blot was used to confirm the production of BCL-6 production by stable infected cells and transiently transfected cells along with COS-7 cells as a positive control. Conclusions demonstrated slight repression that requires further analysis and future use of retroviral infection for such analysis. It does not appear necessary for the BCL-6 to bind at –150 for repression of MCP-1.
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