ABSTRACT
Pir51, a protein of unknown function that interacts with Rad51, was identified in a screen for genes that were highly expressed in aggressive mantle cell lymphoma (MCL) versus indolent small lymphocytic lymphoma (SLL) patient samples. We show that Pir51 is a nuclear protein expressed in a variety of cell types and that its expression is regulated during the cell cycle in a pattern nearly identical to Rad51. Also similar to Rad51, Pir51 levels did not change in response to a variety of DNA damaging agents. siRNA depletion of Pir51 did not reduce homologous recombination repair (HRR), but sensitized cells to mitomycin C (MMC)-induced DNA crosslinking and resulted in elevated levels of double-strand breaks (DSBs) in metaphase chromosome spreads and reduced colony formation. Therefore, Pir51 maintains genomic integrity and potentially connects the early response to DNA crosslinks, orchestrated by the ATR kinase and Fanconi Anemia (FA) proteins, to later stages of Rad51-dependent repair. Our results provide the first example of a Rad51-binding protein that influences DNA crosslink repair without affecting homologous recombination repair.
Subject(s)
Chromosome Breakage/drug effects , DNA-Binding Proteins/genetics , Gene Expression/genetics , Lymphoma/genetics , Mitomycin/pharmacology , Blotting, Northern , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Cell Survival/physiology , DNA Damage/genetics , DNA Repair/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , HeLa Cells , Humans , Lymphoma/metabolism , Mitomycin/metabolism , Mutation/genetics , Protein Binding , RNA, Small Interfering/genetics , RNA-Binding Proteins , Rad51 Recombinase/metabolismABSTRACT
The human, murine, and rat B29 (Ig beta, CD79b) genes are highly conserved in sequence and organization and exhibit strict B cell-specific expression. In the human and rat genomes, the B29 gene is located between the skeletal muscle-specific Na-channel alpha subunit (SCN4A) gene and the pituitary-specific growth hormone (GH-N) gene. The human pituitary-specific GH-N gene is controlled by a tissue-specific locus control region (LCR) located just upstream of the B29 promoter that mediates tissue-specific enhancement, histone acetylation, and an open chromatin conformation across the B29 gene in growth hormone (GH)-expressing pituitary cells. Here we show that B29 mRNA is not detected in a GH-expressing pituitary cell line and that GH-N mRNA is not detected in B cells. This differential expression suggests that the B29 gene is insulated or otherwise protected from the regulatory influences of the closely proximal GH LCR. We searched available sequences upstream of the human, mouse, and rat B29 genes and found a highly conserved sequence that fulfills the criteria recently established for non-coding DNA elements potentially involved in gene control. This B29 conserved sequence (BCS) bound ubiquitously expressed nuclear protein complexes. DNase I protection analysis of the BCS revealed a central 'footprinted' core which was confirmed to bind the multifunctional transcription factor, YY1. However, neither the BCS nor the YY1-binding core motif exhibited silencer or enhancer activity in transient transfections or position-independent insulator activity in enhancer-blocking assays. Thus, the BCS may function as a tissue-specific LCR or position-dependent insulator specifically countering the influences of the 5' GH LCR and controlling B29 gene expression.
Subject(s)
Antigens, CD/genetics , Transcription Factors/metabolism , 5' Flanking Region , Animals , Antigens, CD/metabolism , B-Lymphocytes/physiology , Base Sequence , Binding Sites , CD79 Antigens , Cell Line , Conserved Sequence , Deoxyribonuclease I/metabolism , Enhancer Elements, Genetic , Erythroid-Specific DNA-Binding Factors , Growth Hormone/genetics , Growth Hormone/metabolism , Humans , Locus Control Region , Mice , Molecular Sequence Data , Organ Specificity , Rats , Regulatory Sequences, Nucleic Acid , Silencer Elements, Transcriptional , Transcription Factors/genetics , YY1 Transcription FactorABSTRACT
The B29 (Igbeta) and mb-1 (Igalpha) gene products are B cell-specific essential components of the B cell receptor that are coexpressed at all stages of B cell differentiation, with the exception of plasma cells, which lack mb-1 expression. Transcription of both genes is governed by a similar cassette of interactive transcription factor-binding elements, including octamer motifs, in TATA-less promoters. In this study, we show the B cell-specific B29 gene promoter is transactivated in B and non-B cells by cotransfection with the B cell-specific octamer cofactor gene, Bob1 (OCA-B/OBF-1). The expression of Bob1 is also sufficient to override the silencing effects of the B29 silencer. This indicates that Bob1 plays a critical role in B cell-specific B29 promoter expression. In contrast, coexpression of Bob1 had no effect on mb-1 promoter activity. Bob1 transactivation only occurs with select octamer sequences that have an adenosine at position 5 (ATGCAAAT). The B29 promoter conforms to this consensus octamer motif, while the mb-1 promoter octamer motif does not. Octamer motif swapping between B29 and mb-1 promoters renders B29 unresponsive to Bob1 transactivation and makes mb-1 competent for Bob1 transactivation, thereby indicating that the B29 octamer motif is solely responsible for Bob1 interaction. Additionally, the mb-1 construct containing the B29 octamer motif is expressed in a plasmacytoma cell line, while the wild-type mb-1 promoter is not. Bob1 transactivation of B29 and the lack of this transactivation of mb-1 account for the differential expression of B29 and mb-1 in terminally differentiated plasma cells.
Subject(s)
Antigens, CD/genetics , B-Lymphocytes/metabolism , Promoter Regions, Genetic/immunology , Receptors, Antigen, B-Cell/genetics , Trans-Activators/physiology , Transcriptional Activation/immunology , 3T3 Cells , Animals , Antigens, CD/metabolism , B-Lymphocytes/immunology , CD79 Antigens , Cell Differentiation/genetics , Cell Differentiation/immunology , Consensus Sequence , DNA-Binding Proteins/genetics , Dinucleotide Repeats/immunology , Epitopes, B-Lymphocyte/immunology , Gene Silencing/immunology , Host Cell Factor C1 , Humans , Mice , Octamer Transcription Factor-1 , Plasma Cells/cytology , Plasma Cells/immunology , Plasma Cells/metabolism , Receptors, Antigen, B-Cell/metabolism , Response Elements/immunology , T-Lymphocytes/metabolism , Trans-Activators/biosynthesis , Transcription Factors/genetics , Transfection , Tumor Cells, Cultured , Up-Regulation/immunologyABSTRACT
Interferon-gamma (IFN gamma) exerts diverse responses in B cell development ranging from growth arrest and apoptosis to proliferation and differentiation. IFN gamma stimulates murine 70Z/3 pre-B cells to express surface immunoglobulin (Ig) and this system serves as a useful model for the pre-B to immature B cell transition in B cell development. To analyze this developmental transition, we used a PCR-based subtractive hybridization in combination with miniarray screening to identify differentially-expressed genes in IFN gamma-stimulated compared with unstimulated 70Z/3 pre-B cells. The majority (44%) of the differentially-expressed genes obtained were known IFN gamma-inducible. These included multiple isolates from each of three multi-gene families, including two guanylate-binding protein (47 and 67kDa GBP) families of GTPases and the hematopoietic IFN gamma-inducible nuclear protein family (HIN-200). These multiple isolates of genes comprised the majority of the total isolated and sequenced clones. Other known IFN gamma-induced genes in this group included Ig kappa light chain and Ly-6, as well as genes with functions in antigen processing, cellular regulation, and cytoskeletal organization. Another 36% of the genes identified were previously known, but not known to be IFN gamma-inducible (e.g. pre-B cell enhancing factor, PBEF). The remaining 20% of the IFN gamma-induced isolates did not match entries in Genbank, and thus, may represent novel genes involved in IFN gamma responses and/or in the pre-B to immature B cell transition. Overall, the majority of the individual genes isolated were either not known to be IFN gamma responsive or were not previously known.
