Larry Borish, MD
Genes regulating immunoglobulin synthesis and class switching
Project #1: Functional influences of differential
binding and transcriptional activity with interleukin 10 -571 promoter
Collaborators: J.W. Steinke, L.C. Borish
Use of Cancer Cores: DNA
They have reported a C-to-A SNP at bp -571 in the IL-10 promoter that they and other investigators have linked to breast cancer and HIV survival as well as to incidence and severity of asthma, SLE, and RA. Sp1 was found to bind immediately upstream of this SNP through competition with consensus Sp1 oligonucleotides and Sp1-specific antisera. They did not observe differences in binding affinity of recombinant Sp1 to the C or A forms of the IL-10 promoter. However, when a nuclear extract from an IL-10 producing cell line was used, multimeric complexes were observed which displayed different avidities for the 2 forms of the promoter. Using specific antisera, they found that one of the proteins in complex with Sp1 was Sp3. IL-10 reporter constructs containing the C and A form of the promoter were transfected into the Drosophilia Schneider cell line which lacks endogenous Sp1. Titration of an Sp1 expression vector resulted in decreased transcription from the C form of the promoter, while no inhibition of transcription was observed for the A form of the promoter. Current studies are addressing the role of an ets2 binding site immediately downstream from the SNP and influences in formation of an Sp/Ets/DNA ternary complex.
Project #2: Interaction of the germinal
center-specific GCET (M17) Promoter with Sp1/Sp3
Collaborators: JW Steinke, LC Borish
Use of Cancer Facility: DNA
Antigen-stimulated B lymphocytes undergo additional genetic and phenotypic changes in the context of germinal centers, including affinity maturation of Ig, heavy chain isotype switching, and selection for appropriate Ig specificities. These processes are also directly relevant to processes involved in development of B cell lymphomas and leukemia. To address the identities of factors that control gene expression during humoral immune responses, they examined regulation of the GCET gene, which is specifically expressed in germinal center B cells and whose expression is strongly linked to survival in B cell lymphoma patients. Sequences that constitute a functional promoter in GCET-expressing cells were determined. They identified the specific binding of Sp1/Sp3 to the sequence CCTCCT at position -40 to -45. Transfection of the GCET promoter into Drosophilia Schneider cells, which lack endogenous Sp1 transcription factor confirmed a role for Sp in the promoter of this TATAA-less gene. In addition, they have identified a role for pax5 in regulation of GCET, which extends to germinal centers a role for this B cell-specific "master regulator."
Project #3: Different immune signaling through the
cysteinyl leukotriene G-protein coupled receptors
Collaborators: J.W. Steinke, L.C. Borish, K. Ravichandran
Use of Cancer Center Cores: DNA, FACS
They are investigating unique roles of the 2 CysLT receptors in inflammatory disorders. They have hypothesized that CysLT1 receptors may be important to bronchospasm and immune cell trafficking whereas CysLT2 are more important in pathways leading to tissue damage, fibrosis, and remodeling. Current studies are addressing unique CysLT1 and CysLT2-receptor mediated tyrosine phosphorylation pathways in leukotriene-activated eosinophil lines (HL60 clone 15).