FAR UPSTREAM REGIONS OF CLASS-II MHC EA ARE NECESSARY FOR POSITION-INDEPENDENT, COPY-DEPENDENT EXPRESSION OF EA TRANSGENE

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