Functional analysis of cis‐linked regulatory sequences in the HLA DRA promoter by transcription in vitro

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Abstract
Two consensus sequences, called × and Y boxes, capable of binding nuclear proteins and regulating expression in B cells have been defined within the immediate upstream region of major histocompatibility complex (MHC) class II promoters. Unlike other class II promoters, the HLA‐DR a (DRA) promoter also contains one element identical to the “octamer” motif of immunoglobulin variable region promoters that is responsible for B cell‐specific transcription. This “octamer” in the context of DRA appears capable of binding both the ubiquitous (OTF‐I) and lymphoid‐specific (OTF‐2) “octamer” binding proteins, but at least one other distinct “octamer” complex was found. In order to characterize the function of cis‐acting elements, we have developed an in vitro system in which a DRA promoter construct is transcribed more efficiently in extracts from B cells than in extracts from class II‐negative HeLa cells. 5′ deletion constructs which lacked the Y box, but retained the “octamer” the Y box reduced transcription by 95%. Using supercoiled, but not linear templates, we observed differences in transcription efficiencies from templates lacking or disrupting the × consensus element that reflect effects of random replacement of × box sequences in transient expression assays. Demonstration of the complete dependence on the Y box in this system suggests that, despite its demonstrated importance in the DRA promoter, the DRA “octamer” does not utilize OTF‐2 in a manner analogous to immunoglobulin promoters in B cells.

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