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Repression of the PDCD2 Gene by BCL6 and the Implications for the Pathogenesis of Human B and T Cell Lymphomas

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Repression of the PDCD2 Gene by BCL6 and the Implications for the Pathogenesis of Human B and T Cell Lymphomas Repression of the PDCD2 gene by BCL6 and the implications for the pathogenesis of human B and T cell lymphomas Beverly W. Baron*†, Nancy Zeleznik-Le‡, Miriam J. Baron§, Catherine Theisler‡, Dezheng Huo¶, Matthew D. Krasowski*ሻ, Michael J. Thirman**, Rebecca M. Baron§, and Joseph M. Baron** Departments of *Pathology, ¶Health Studies, and **Medicine, University of Chicago, Chicago, IL 60637; ‡Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL 60153; and §Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 Communicated by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, March 2, 2007 (received for review September 26, 2006) The human BCL6 gene on chromosome 3 band q27, which encodes specifically directed against the BCL6 and PDCD2 proteins a transcriptional repressor, is implicated in the pathogenesis of revealed a differential staining pattern of these proteins with an human lymphomas, especially the diffuse large B-cell type. We inverse relationship between the localization of their expression. previously identified the human PDCD2 (programmed cell death-2) Analysis of the sequences flanking the first exon of PDCD2 gene as a target of BCL6 repression. PDCD2 encodes a protein that showed an exact match to a previously identified high-affinity is expressed in many human tissues, including lymphocytes, and is BCL6 binding site located Ϸ3.4 kb upstream of exon 1 (18). known to interact with corepressor complexes. We now show that However, a direct relationship between these genes has not been BCL6 can bind directly to the PDCD2 promoter, repressing its demonstrated previously. transcription. Knockdown of endogenous BCL6 in a human B cell The role of BCL6 as a transcriptional repressor in lym- lymphoma line by introduction of small interfering RNA duplexes phomagenesis requires detailed study of its effect on its target increases PDCD2 protein expression. Furthermore, there is an promoters. We now report that (i) BCL6 binds to the PDCD2 inverse relationship between the expression levels of the BCL6 and promoter and functionally represses PDCD2 expression at the PDCD2 proteins in the lymphoid tissues of mice overexpressing transcriptional level, (ii) direct silencing of BCL6 results in human BCL6 (high BCL6 levels, minimal PDCD2) and controls (minimal BCL6, high PDCD2) as well as in tissues examined from augmented PDCD2 expression and likely significant down- some human B and T cell lymphomas. These data confirm PDCD2 as stream effects thereof, and (iii) an inverse relationship between a target of BCL6 and support the concept that repression of PDCD2 BCL6 and PDCD2 expression is present in the lymphoid tissues by BCL6 is likely important in the pathogenesis of certain human of BCL6 transgenic mice and, more importantly, in 10 human lymphomas. lymphoma patient samples. human lymphomas ͉ target of BCL6 Results The BCL6 Protein Binds to the PDCD2 Promoter. In vitro binding CL6, a gene on chromosome 3, band q27, encodes a nuclear studies with EMSAs revealed that the HA-tagged BCL6 protein Bzinc finger protein that is a transcriptional repressor (1–3). binds specifically to the high-affinity consensus site we identified This protein is expressed at high levels in human lymph node in the PDCD2 promoter [GGACCTACCCTTCTAGGAA- germinal center B cells, most cortical thymocytes, and some AAAACCATCC; the 9- of 9-nt exact match is italicized (18)] human B and T cell lymphomas (4). The BCL6 gene was (Fig. 1a). An excess of nonradioactive double-stranded oligomer identified (5–7) through its involvement in chromosomal trans- containing the BCL6 consensus site competed for binding with locations that occur in Ϸ40% of diffuse large-cell B cell lym- the radioactive double-stranded oligomer, and binding was phomas. In other lymphomas, mutations occur 5Ј to the BCL6 reduced by the addition of antibodies specifically directed coding region (8). The BCL6 protein binds DNA in a sequence- against BCL6 and HA; in contrast, an unrelated antibody specific fashion through its C-terminal zinc finger region (9, 10). (CD45RB; Lab Vision, Fremont, CA) and PBS did not affect It conveys transcriptional repression through an N-terminal binding. The EMSA was performed additionally with recombi- POZ domain and a second domain that is more centrally located nant BCL6 protein and vector not containing BCL6 (control) (1–3, 11) and interacts with a number of corepressors (12–14). generated by in vitro transcription/translation (Fig. 1b). Whereas It is thought that the BCL6-repressive effects are mediated the transcription/translation BCL6 zinc-finger protein binds to through multiprotein repression complexes with histone the 32P-labeled double-stranded oligomers containing the BCL6 deacetylase activity. A peptide that specifically binds BCL6 and consensus binding site in the PDCD2 promoter, the vector does blocks corepressor recruitment leads to apoptosis and cell-cycle not. These data show that the binding between BCL6 and its arrest of BCL6-positive lymphoma cells (15). Mice overexpress- consensus binding site in the PDCD2 promoter is specific. We ing human (16) or murine (17) BCL6 develop lymphomas. and others (figure 2 in ref. 3 and figure 5B in ref. 20) have We previously generated a dominant-negative cell system (18) previously demonstrated a reduction in binding with the use of with the use of a construct expressing the BCL6 zinc fingers, HA antibodies. Reduction in binding without a supershift may which compete with the binding of endogenous BCL6 in BJAB cells (an Epstein–Barr virus-negative Burkitt lymphoma cell line expressing high levels of BCL6) (19). Because BCL6 is a Author contributions: B.W.B., N.Z.-L., M.D.K., M.J.T., and R.M.B. designed research; B.W.B., repressor, competition for binding of the full-length endogenous N.Z.-L., C.T., and R.M.B. performed research; J.M.B. contributed new reagents/analytic BCL6 protein by the exogenously transfected zinc fingers results tools; B.W.B., N.Z.-L., M.J.B., C.T., D.H., R.M.B., and J.M.B. analyzed data; and B.W.B. wrote in up-regulation of BCL6 target genes. We used subtractive the paper. hybridization techniques to amplify differentially expressed se- The authors declare no conflict of interest. quences. These studies led to the identification of the PDCD2 †To whom correspondence should be addressed. E-mail: [email protected]. (programmed cell death-2) gene as a target of BCL6. Immuno- ࿣Present address: Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261. CELL BIOLOGY histochemistry performed on human tonsil with antibodies © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701770104 PNAS ͉ May 1, 2007 ͉ vol. 104 ͉ no. 18 ͉ 7449–7454 Downloaded by guest on September 24, 2021 Fig. 1. EMSA. (a) The BCL6 protein, which was prepared from nuclear extracts of COS cells transfected with a full-length BCL6 expression construct, binds specifically to the PDCD2 promoter. Lane 1, nonprotein control (no shifted bands). The BCL6 protein binds specifically to the PDCD2 promoter sequences containing the BCL6 binding site because the shifted band (lane 2, arrow) is competed with a 310-fold molar excess of the same nonradiolabeled double-stranded oligonucleotide (lane 3), and its binding is reduced by the addition of antibodies to BCL6 (lane 4). Lane 4 is from the same gel as lanes 1–3. Lane 5, nonprotein control (no shifted bands). Lane 6, 32P-labeled double- stranded PDCD2 promoter oligonucleotides. The heavy shifted band (arrow) is reduced by the addition of antibodies to the HA tag in the BCL6 expression construct (lane 7) but not by an unrelated antibody (lane 8) or by PBS (lane 9). (b) The in vitro transcribed/translated BCL6 zinc-finger protein binds to the PDCD2 promoter sequences containing the BCL6 binding site (arrow), whereas the vector control (V) does not bind. Fig. 3. The BCL6 protein represses transcription from the PDCD2 promoter (transfection assays). (a) Relative luciferase levels in NIH 3T3 cells reveal be caused by antibody interfering with the DNA binding of the significant repression (P Ͻ 0.0005) of PDCD2 promoter activity by the full- protein. length BCL6 protein (mean Ϯ SE, 0.63 Ϯ 0.06) as compared with a truncated Furthermore, ChIP assays confirmed that in a B cell lym- control that cannot bind DNA because it lacks the BCL6 zinc fingers, which are phoma line, endogenous BCL6 was bound to the region in needed to bind DNA. For purposes of comparison, the data were normalized PDCD2 containing the high-affinity BCL6 binding site, because with results for the control designated as 1. (b) Similarly, in HeLa cells, PDCD2 promoter activity (mean Ϯ SD, 3,327 Ϯ 104 in the absence of BCL6) was DNA from this region was enriched in chromatin immunopre- repressed significantly (P ϭ 0.01) by the full-length BCL6 protein (mean Ϯ SD, 2,407 Ϯ 584). cipitated with anti-BCL6 (Fig. 2a). This genomic region was not enriched in material immunoprecipitated with anti-HA or in the absence of antibody (controls). No binding of BCL6 to the coding region of PDCD2, which lacks a putative binding site, was observed (Fig. 2b). Similarly, BCL6 does not bind to the GAPDH gene (data not shown), confirming the specificity of BCL6 binding to the PDCD2 promoter. The BCL6 Protein Represses Transcription From the PDCD2 Promoter. Transient transfections were performed in NIH 3T3 cells [which express BCL6 only weakly (21) and have minimal levels of PDCD2 (unpublished data)]. Relative luciferase levels, depicted in Fig. 3a, reveal significant repression (P Ͻ 0.0005) of PDCD2 promoter activity by the full-length BCL6 protein (mean Ϯ SE, 0.63 Ϯ 0.06) as compared with a truncated control that cannot bind DNA (for purposes of comparison, the absolute value of the control was set at 1).
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