Analysis of differential HLA-DQB expression in autologous B cell lines

Abstract

Class II major histocompatibility complex genes are differentially expressed during cellular activation and differentiation, often in a locus-specific manner. We investigated the differential expression of the HLA-DQB gene, using B cell lines LAZ221 and LAZ388: LAZ221, derived from an early B cell leukemia, expresses HLA-DR but not HLA-DQ: LAZ388, the autologous Epstein-Barr virus-transformed B cell line, expresses both DR and DQ. Transfection experiments demonstrate differential function of class II gene upstream regulatory regions in the two lines, which correlates with differential class II gene expression. Using gel retardation and DNase I footprint assays, we demonstrate that absence of DQB gene expression is associated with characteristic nuclear protein-binding interactions in the proximal DQB gene upstream regulatory region. These interactions are visualized as DNA-protein complexes that are seen with nuclear proteins from the DQ-negative cell line, LAZ221, and involve consensus promoter Y box and W box elements, as well as novel upstream sites. Transcriptional regulatory proteins that differ in these autologous B cell lines may be stage-specific factors involved in the developmental regulation of HLA genes.

DNA methylation dysregulates and silences the HLA-DQ locus by altering chromatin architecture

“…Previous analyses demonstrated differential expression of HLA-DR and HLA-DQ surface protein levels between the Laz221 and Laz388 cell lines 24, 25. This data is recapitulated in Figure 1A, which shows two distinct expression profiles for these cells.…”

“…Laz221 cells were similar in phenotype to the ALL cells of the patient and were considered to represent null cells at the time. Later Laz221 cells were determined to be most similar to a pre-B lymphocyte and were thought to arise from these cells 24, 25. The cell line Laz388 was established from the same patient by transforming with Epstein-Barr-virus 23, 26 and represented a normal peripheral B cell.…”

“…The cell line Laz388 was established from the same patient by transforming with Epstein-Barr-virus 23, 26 and represented a normal peripheral B cell. One curious difference between the cells was that whereas Laz388 expressed both HLA-DR and HLA-DQ proteins, Laz221 only expressed the HLA-DR proteins 24, 25. Analysis of the cells showed that the HLA-DR genes were expressed in Laz221 but neither HLA-DQA or HLA-DQB was expressed 24.…”

“…One curious difference between the cells was that whereas Laz388 expressed both HLA-DR and HLA-DQ proteins, Laz221 only expressed the HLA-DR proteins 24, 25. Analysis of the cells showed that the HLA-DR genes were expressed in Laz221 but neither HLA-DQA or HLA-DQB was expressed 24. Examination of the cis -regulatory elements by transfection of DNA reporter constructs into each of the cell lines and the use of EMSAs suggested that the HLA-DQB1 gene was regulated differently and that the Laz221 cell line was lacking a critical component 24, 27.…”

“…Analysis of the cells showed that the HLA-DR genes were expressed in Laz221 but neither HLA-DQA or HLA-DQB was expressed 24. Examination of the cis -regulatory elements by transfection of DNA reporter constructs into each of the cell lines and the use of EMSAs suggested that the HLA-DQB1 gene was regulated differently and that the Laz221 cell line was lacking a critical component 24, 27. Because Laz221 expressed HLA-DR molecules at normal levels, this meant that each of the MHC-II specific transcription factors RFX5, RFX-B, RFXAP, and CIITA were present and fully active and that some other aspect of the system was defective.…”