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. 2002 Mar 5;99(5):2860-5.
doi: 10.1073/pnas.042702599. Epub 2002 Feb 19.

The human programmed cell death-2 (PDCD2) gene is a target of BCL6 repression: implications for a role of BCL6 in the down-regulation of apoptosis

Beverly W Baron  1 , John Anastasi, Michael J Thirman, Yoichi Furukawa, Scott Fears, David C Kim, Federico Simone, Mark Birkenbach, Anthony Montag, Annamma Sadhu, Nancy Zeleznik-Le, Timothy W McKeithan
Affiliations

The human programmed cell death-2 (PDCD2) gene is a target of BCL6 repression: implications for a role of BCL6 in the down-regulation of apoptosis

Beverly W Baron et al. Proc Natl Acad Sci U S A. .

Abstract

BCL6, a gene on chromosome 3 band q27, encodes a Kruppel-type zinc finger transcriptional repressor. Rearrangements of this gene are frequent in various kinds of lymphomas, particularly of the large-cell B-cell type. The BCL6 nuclear phosphoprotein is expressed in a variety of tissues and is up-regulated particularly in lymph node germinal centers. The zinc fingers of BCL6 bind DNA in a sequence-specific manner. To identify targets of the BCL6 repressive effects, we used a VP16-BCL6 fusion protein containing the zinc fingers but devoid of the repressor domains to compete with the binding of endogenous BCL6 in a transiently transfected B-cell line and then performed subtractive hybridization by using a method to selectively amplify sequences that are differentially expressed. We found that the programmed cell death-2 (PDCD2) gene is a target of BCL6 repression. This gene is the human homolog of Rp8, a rat gene associated with programmed cell death in thymocytes. Immunohistochemistry reveals the anticipated inverse relationship between BCL6 and PDCD2 expression in human tonsil. PDCD2 is detectable in cells of the germinal center in areas where there is less BCL6 expression as well as in the mantle zone, where there is little or no BCL6 expression. These results raise the possibility that BCL6 may regulate apoptosis by means of its repressive effects on PDCD2. BCL6 deregulation may lead to persistent down-regulation of PDCD2, reduced apoptosis, and, as a consequence, accumulation of BCL6-containing lymphoma cells.

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Figures

Figure 1
Figure 1
RT-PCR of RNA from VP16-BCL6ZF-Transfected BJAB Cells. Lane 1, 1 Kb-DNA Ladder; lane 2, RT-PCR with β-actin primers that amplify 285 bp; lane 3, negative control, same primers, no RT; lane 4, negative control, same primers (water template); lane 5, RT-PCR with primers across the VP16-BCL6ZF junction that amplify 370 bp of cDNA; lane 6, negative control, VP16-BCL6ZF primers, no RT; lane 7, negative control, same primers (water template).
Figure 2
Figure 2
EMSA. Lane 1, nonprotein control (no shifted bands). The VP16-BCL6ZF protein binds specifically to the BCL6 consensus site, because the heavy shifted band (lane 2, bottom arrow) is displaced by mixing the radiolabeled double-stranded oligomer containing the BCL6 binding site with a 338-fold molar excess of the same nonradiolabeled double-stranded oligo (lane 3), is not displaced by mixing the hot probe with a 715-fold molar excess of cold mutant double-stranded oligomer containing point mutations that block BCL6 binding (lane 4), and its binding is reduced (bottom arrow) and supershifted (top arrow) by addition of polyclonal rabbit antibodies to the BCL6 carboxy terminus (lane 5) and polyclonal antibodies recognizing the HA epitope tag (lane 6). Nuclear extracts of untransfected COS cells do not bind specifically to the probe (lane 7). A nonspecific cold DNA competitor was added to each reaction. The central arrow depicts a nonspecifically binding band.
Figure 3
Figure 3
The upper panel is an autoradiograph of a Northern blot prepared from total RNA of BJAB cells transiently transfected with the study (S) expression construct (contains VP16-BCL6ZF-HA) or the control (C, contains vector with VP16-HA); RNA sizes (kb) are indicated on the left. (Upper) Hybridization with a cDNA fragment of PDCD2 obtained from subtractive hybridization; its ≈1.5-kb transcript is noted. The blot was stripped and rehybridized with a human β-actin probe (Lower). Differential expression of PDCD2 normalized to β-actin is 5-fold.
Figure 4
Figure 4
Western blots prepared from whole-cell extracts of human 293 cells transfected with a Flag-tagged PDCD2 expression construct or an unrelated Flag-tagged expression construct, EAF1 (control). (Left) Western blot incubated with anti-Flag; the expected ≈43-kDa band is present in the EAF1 extract. A band at ≈45 kDa is recognized by anti-Flag in the extract from the PDCD2-transfected cells. (Right) The same ≈45-kDa band is recognized by anti-PDCD2, but, as expected, no band is recognized by this antibody in the EAF1-transfected cells.
Figure 5
Figure 5
Immunohistochemistry. Frozen sections (4 μm) of human tonsil were stained with a mouse monoclonal antibody to BCL6 (Left) or rabbit polyclonal antibodies to PDCD2 (Right). The anticipated inverse relationship between BCL6 and PDCD2 localization is noted. The antibody to BCL6 stains nuclei of lymphocytes in the germinal center most heavily in larger cells (centroblasts, toward top of Left and detail in top Inset—dark brown nuclei), whereas PDCD2 localizes in the cytoplasm of germinal center cells (toward bottom of Right and detail in bottom Inset) in areas where there is less BCL6 staining (Left, bottom of germinal center and detail in bottom Inset). There is little PDCD2 staining (detail, Right, top Inset) in areas where BCL6 stains heavily (Left, top Inset). PDCD2 is also noted in a number of follicular mantle cells which do not express BCL6.
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