BLIMP1 Downregulates Expression of, LMO2 and HGAL, Proteins Highly Expressed In Germinal Center B Cells and Germinal Center-Derived Lymphomas

Abstract 4133

Human germinal center–associated lymphoma (HGAL) and LIM domain only-2 (LMO2) are proteins highly expressed in germinal center (GC) B lymphocytes. HGAL and LMO2 are also expressed in GC-derived lymphomas and distinguish biologically distinct subgroups of diffuse large B-cell lymphomas (DLBCL) associated with improved survival. HGAL is involved in motility regulation of both normal and malignant B cells, while the function of LMO2 in GC B cells and lymphomas is unknown. However, LMO2, functioning as a transcription factor, is frequently activated in childhood T-cell acute lymphoblastic leukemias by chromosomal rearrangement or retroviral integration inducing thymocyte self renewal (McCormack, 2010) leading to oncogenic transformation. LMO2 is also necessary for embryonic erythropoiesis and adult hematopoiesis.

PRDM1/BLIMP1 encodes a zinc finger transcriptional repressor that is expressed in a subset of GC B cells and in all plasma cells. It is a master regulator of terminal B cell differentiation to plasma cells. BLIMP1 may also function as a tumor suppressor in the pathogenesis of DLBCL, where it is frequently inactivated by mutations and deletions. Blimp1 promotes plasma cell differentiation and exerts its tumor suppressive effects by inhibiting the expression of genes important for GC B cell functions like c-myc, CIITA, PAX5, Bcl6, Spi-B, and ID-3 (Shaffer, 2002). Here, we demonstrate that Blimp1 also inhibits LMO2 and HGAL expression by direct binding to their promotors.

Transient over-expression of BLIMP1 in B lymphoma cell lines results in a decrease in RNA and protein levels of HGAL and LMO2, as assessed by quantitative real time PCR and immunoblotting. To confirm a direct effect of BLIMP1 on expression of the HGAL and LMO2 genes, we have cloned the corresponding 2046 bp and 2549 bp DNA promoter sequences containing putative binding sites of BLIMP1 into luciferase reporter constructs. Over-expression of BLIMP1 in HeLa, VAL and CA-46 cell lines induces a statistically significant decrease in the promoter activity of both HGAL and LMO2. Site-specific mutagenesis of the BLIMP binding sites partially reverses the inhibitory effect of the BLIMP1 protein on the promoter activity, thus confirming specificity of the observed BLIMP1 inhibitory effects. To further demonstrate existence of the BLIMP1 inhibitory effect in a physiological context, we demonstrated in vivo binding of the endogenous BLIMP1 protein to HGAL and LMO2 promoters in plasma cells lines by chromatin immunoprecipitation assays. These findings demonstrate that Blimp1 is a physiological transcriptional repressor of the expression of LMO2 and HGAL genes. This inhibitory effect may contribute to the disappearance of HGAL and LMO2 expression upon differentiation of GC B cell to plasma cells and may also lead to absence of HGAL and LMO2 expression in post-GC lymphoid tumors. Additional studies are necessary to demonstrate whether inhibition of LMO2 and HGAL expression is necessary for oncogene suppressor functions of the BLIMP1 in DLBCL pathogenesis.