The Relationship between RUVBL1 (Pontin, TIP49, NMP238) and BCL6 in Benign and Malignant Human Lymphoid Tissues

Abstract

The human BCL6 gene, which is involved in the pathogenesis of certain human lymphomas, encodes a transcriptional repressor that is needed for germinal center B cell development and T follicular helper cell differentiation. Our goal was to identify BCL6 target genes using a cell system in which BCL6 repressive effects are inhibited followed by subtractive hybridization, and we detected the RUVBL1 (Pontin, TIP49) gene as a potential target of BCL6 repression. Here we show that the BCL6 protein significantly represses RUVBL1 transcription (6.8-fold). Knockdown of endogenous BCL6 in a human B cell lymphoma line leads to significant upregulation of RUVBL1, and there is an inverse expression pattern between the BCL6 and RUVBL1 proteins in certain human lymphomas. RUVBL1 is part of the AAA+ superfamily and participates in multiple processes, including gene transcription regulation, chromatin remodeling, and DNA repair, which, if dysregulated, may promote lymphoma development. A further understanding of the relationship between RUVBL1 and BCL6 should improve our understanding of the pathogenesis of human lymphomas.

Keywords: BCL6 target; NMP238; Pontin; RUVBL1; TIP49; lymphomagenesis.

Fig. 1

Northern blotting confirms differential expression of RUVBL1. (A) Representative autoradiograph of a Northern blot made from total RNA of BJAB cells (human B cell lymphoma line) transiently transfected with an expression construct (S, study) containing the BCL6 fingers or the vector (control, C) in which the S construct was cloned. Top: Hybridization with a cDNA fragment of RUVBL1 obtained from subtractive hybridization shows its 1.8 kb transcript. Bottom: The blot was stripped and hybridized with a human β-actin probe. (B) Densitometry data showing differential expression of RUVBL1 normalized to β-actin. For eight Northern blots, differential expression of RUVBL1 (S lane) as compared with the control (C) is significant by unpaired t test; mean±SEM, 2.593±0.497 (range 1.18–5.72)-fold (p<0.006). The data were normalized with results for the control designated as 1.

Fig. 2

The BCL6 protein represses transcription from the RUVBL1 promoter (transfection assays). Relative luciferase levels in 293 T cells show that RUVBL1 promoter activity is significantly repressed (6.8-fold, p<0.0001) by the full-length BCL6 protein (mean±SEM=0.146±0.059) as compared with a truncated control that lacks the BCL6 zinc fingers and thus cannot bind DNA. The data were normalized with results for the control set at 1.

Fig. 3

Knockdown of BCL6 protein levels by siRNA increases RUVBL1 protein expression. (A) Representative Western blot depicting BCL6 (95 kDa), RUVBL1 (50 kDa), and β-actin protein expression in BJAB cells 45 h after transfection with siRNA duplexes targeting BCL6 [C, control siRNA; S, study (BCL6) siRNA]. All bands are from the same blot. (B) The graph indicates the relative protein expression of BCL6 and RUVBL1 after normalization for β-actin expression. BCL6 protein expression (left): mean±SEM for control siRNA 1.783±0.556, and for BCL6 siRNA 0.595±0.216, p=0.064. RUVBL1 protein expression (right): mean±SEM for control siRNA 1.146±0.205, and for BCL6 siRNA 2.229±0.47, p<0.05.

Fig. 4

ChIP assay. (A) RUVBL1 promoter region containing the high-affinity BCL6 binding site (0.705 kb). In BJAB cells BCL6 is bound (perhaps as part of a complex) to the region of the RUVBL1 promoter containing the putative BCL6 binding site (arrow) because DNA from this region is enriched in chromatin immunoprecipitated with anti-BCL6 (lane 5). Controls – no antibody (lane 1) and unrelated antibody, anti-rabbit IgG (lane 2) – do not enrich this genomic region. The positive (+) control is genomic DNA amplified with the same primers (lane 6). Lane 4 is 1 Kb Plus DNA Ladder (Invitrogen™, Life Technologies, Grand Island, NY); lane 7 is input. (B) RUVBL1 coding region (0.162 kb, arrow). Binding of BCL6 to a part of the coding region of RUVBL1 is not observed (lane 2) because there is no putative binding site. Input (lane 4) and the positive (+) control, genomic DNA amplified with the same primers (lane 5), show the anticipated 162 bp product (arrow). Lane 3 is 1 Kb Plus DNA Ladder (Invitrogen™).

Fig. 5

Immunohistochemistry of representative randomly chosen human lymphomas stained for BCL6 and RUVBL1. The H&E stain shows effacement of normal tissue architecture by malignant cells. Panel 1: T cell tumor, BCL6 negative, RUVBL1 positive. Panel 2: B cell lymphoma, BCL6 negative area, where RUVBL1 is positive. Panel 3: Same B cell lymphoma as in panel 2, BCL6 positive area, where RUVBL1 staining is negative. Panel 4: Germinal center of human tonsil; BCL6 and RUVBL1 are both expressed within the germinal center, but many more lymphocytes outside the germinal center express RUVBL1 as compared with BCL6. The bars in the lower right photos indicate 20 µm for each image in the corresponding panel.