Examination of the molecular basis for the lack of alphaB-crystallin expression in L929 cells.

Abstract

We have previously shown that murine L929 cells do not express the small heat shock protein alphaB-crystallin upon exposure to thermal stress (Mol Cell Biochem 155: 51-60, 1996). In these studies, we demonstrate that L929 cells also fail to express alphaB-crystallin upon exposure dexamethasone, whereas NIH 3T3 and Swiss 3T3 murine cells exhibit alphaB-crystallin expression under identical conditions. Mobility shift assays demonstrated heat-inducible binding, presumably by heat shock factor(s), to an alphaB-crystallin heat shock element (HSE) oligomeric sequence in total cellular extracts from L929 cells. Transient transfection of a plasmid containing the alphaB-crystallin promoter linked to a CAT reporter gene exhibited heat-inducible expression in L929 cells. In addition, L929 cells stably transfected with a plasmid containing the complete alphaB-crystallin gene showed expression of this gene following heat shock. The presence of the endogenous alphaB-crystallin gene was detected by Southern blot hybridization of genomic L929 DNA, and sequence analysis revealed identical nucleotide structure to published murine sequences throughout the entire promoter. Treatment of L929 cells with 5-azacytidine enabled heat-inducible expression of alphaB-crystallin from the endogenous gene, however, methylation of the putative heat shock element (HSE) and flanking promoter sequences of L929 cell genomic DNA was not detected. In vivo genomic footprinting demonstrated constitutive binding to the endogenous HSE of the alphaB-crystallin promoter in L929, L929/alphaB-crystallin transfectant cells, and Swiss 3T3 cells during unstressed and heat stressed conditions. Therefore, the genomic alphaB-crystallin HSE region in L929 cells appears to be available for binding of putative transcription factors, but methylation in other regions of the gene or genome repress the expression of alphaB-crystallin in L929 cells. In vitro culture of L929 cells appears to have rendered the alphaB-crystallin gene loci inactive through methylation, thus providing a unique system by which to study the function of transfected small heat shock proteins.