EXAMINATION OF THE MOLECULAR-BASIS FOR THE LACK OF ALPHA-B-CRYSTALLINEXPRESSION IN L929 CELLS

We have previously shown that murine L929 cells do not express the sma ll heat shock protein alpha B-crystallin upon exposure to thermal stre ss (Mol Cell Biochem 155: 51-60, 1996). In these studies, we demonstra te that L929 cells also fail to express alpha B-crystallin upon exposu re dexamethasone, whereas NIH 3T3 and Swiss 3T3 murine cells exhibit a lpha B-crystallin expression under identical conditions. Mobility shif t assays demonstrated heat-inducible binding, presumably by heat shock factor(s), to an alpha B-crystallin heat shock element (HSE) oligomer ic sequence in total cellular extracts from L929 cells. Transient tran sfection of a plasmid containing the alpha B-crystallin promoter linke d to a CAT reporter gene exhibited heat-inducible expression in L929 c ells. In addition, L929 cells stably transfected with a plasmid contai ning the complete alpha B-crystallin gene showed expression of this ge ne following heat shock. The presence of the endogenous alpha B-crysta llin 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 o f L929 cells with 5-azacytidine enabled heat-inducible expression of a lpha B-crystallin from the endogenous gene, however, methylation of th e 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 alpha B -crystallin promoter in L929, L929/alpha B-crystallin transfectant cel ls, and Swiss 3T3 cells during unstressed and heat stressed conditions . Therefore, the genomic alpha B-crystallin HSE region in L929 cells a ppears to be available for binding of putative transcription factors, but methylation in other regions of the gene or genome repress the exp ression of alpha B-crystallin in L929 cells. In vitro culture of L929 cells appears to have rendered the alpha B-crystallin gene loci inacti ve through methylation, thus providing a unique system by which to stu dy the function of transfected small heat shock proteins.