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The chromatin upstream of the class II MHC Ea gene contains specific, DNase I hypersensitive (DH) sites (groups I-V), overlapping and extending the promoter proximal and distal control regions. To determine whether the Ea DH groups I-V define a functionally important chromatin domain or locus control region (LCR), we have used wild type Ead gene constructs to generate transgenic mouse lines from strains that do not express an endogenous Ea gene product. Constructs contained either DH groups I-V 'Longs' or DH groups I-II 'Shorts', of the hypersensitive sites defined within 20 kb 5' of Ea. We show that position-independent, copy number-dependent expression of the Ead gene occurs only with the Long construct (8/8 transgenic mouse lines, over a range of copy numbers, 1-30 copies); in contrast, the Short constructs are subject to position-dependent effects. This suggests that the region delineated by Ea DH groups I-II is necessary but not sufficient as an LCR, which requires the presence of the upstream regions containing DH III-V for complete position-independent, copy number-dependent expression. These results introduce an immunologically-important, putative LCR which can be used to target genes to cells of the B cell lineage, as well as to other class II MHC expressing cells, and highlight the importance of chromatin structure analysis as a means to locate DNA regions of regulatory interest which are dispersed over a large distance.

Far upstream regions of class II MHC Ea are necessary for position-independent, copy-dependent expression of Ea transgene