No abstract

The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC-box as an essential element for PRDM1 expression. This GC-box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC-box.Key words: B cell differentiation Á Human Á PRDM1 Á Transcription factors Supporting Information available online IntroductionPlasma cells (PC), i.e. terminally differentiated B lymphocytes in a post-mitotic state, are immunoglobulin (Ig)-secreting cells playing a central role in the antigen-specific immune response. The plasmacytic differentiation process is regulated by the "turn off" and "turn on" of many specific transcription factors (TF), which are now starting to be elucidated [1][2][3][4]. One of these, the human positive regulatory domain containing 1 with zinc-finger domain (PRDM1), is considered as a key factor that promotes the nonproliferative and differentiated stage of PC [4, 5]. The PRDM1 factor was initially identified as a 98-kDa DNA-binding protein that contains five zinc-finger domains and acts as a repressor of interferon-b gene expression [6]. Its murine homolog, initially called B lymphocyte-induced maturation protein-1 (Blimp-1), was isolated as a mouse gene that is induced during B lymphocyte maturation [7]. It is expressed at high levels only in late B cells and PC, and the ectopic overexpression of PRDM1 is sufficient to drive B lymphocytes to differentiate into PC [7]. Moreover, its expression is required for the formation of Ig-secreting cells [8] and for the maintenance of long-lived PC in bone marrow [9].MYC Here we have defined the minimal promoter for human PRDM1 transcription enclosing a GC-rich element necessary for basal transcription. Moreover, this GC-rich element contains an SP1/early growth response 1 (EGR-1) overlapping binding site to which the regulatory factors SP1, SP3 and EGR-1 can bind, which activate the PRDM1 transcription. Results and discussionStructure of the 5 0 -flanking region and initiation sites of the human PRDM1 gene To obtain insight into the regulation of the human PRDM1 gene and b...

PCs are responsible for the production and secretion of antibodies, the effector molecules of the humoral immune response. The molecular mechanisms responsible for vesicle docking and secretion implicated in the antibody-secretion process are not well-known, as they have not been studied, but it is known that SNARE proteins are responsible for many membrane-fusion processes in the cell. We show here that freshly isolated human colon LP-PCs and T-PCs from MM-PC patients and the U266 cell line, as a model for PC secretion, contain a set of these proteins. SNAP23, STX3, and STX4 were localized mainly in the plasma membrane of PCs, and interactions of SNAP23 with STX3 and with STX4 were proven by IP. Interaction between SNAP23 and STX4 was also confirmed in situ. With the use of siRNA, as well as shRNA, the functional role of SNAP23, STX3, and STX4 in antibody secretion was also examined. The findings demonstrate that in addition to SNAP23, STX4 is implicated in the antibody secretion by a myeloma cell line and by normal human colon LP-PCs.