Regulation of the Myeloid-Cell-Expressed Human gp91-phox Gene As Studied by Transfer of Yeast Artificial Chromosome Clones into Embryonic Stem Cells: Suppression of a Variegated Cellular Pattern of Expression Requires a Full Complement of Distant cis Elements

Abstract

Identifying the full repertoire of cis elements required for gene expression in mammalian cells (or animals) is challenging, given the moderate sizes of many loci. To study how the human gp91-phox gene is expressed specifically in myeloid hematopoietic cells, we introduced yeast artificial chromosome (YAC) clones and derivatives generated in yeast into mouse embryonic stem cells competent to differentiate to myeloid cells in vitro or into mouse chimeras. Fully appropriate regulation was recapitulated with a 130-kb YAC containing 60 and 30 kb of 5′ and 3′ flanking sequences, respectively. Immunodetection of human gp91-phox protein revealed uniform expression in individual myeloid cells. The removal of upstream sequences led to decreased overall expression which reflected largely a variegated pattern of expression, such that cells were either “on” or “off”, rather than pancellular loss of expression. The proportion of clones displaying marked variegation increased with progressive deletion. DNase I mapping of chromatin identified two hypersensitive clusters, consistent with the presence of multiple regulatory elements. Our findings point to cooperative interactions of complex regulatory elements and suggest that the presence of an incomplete set of elements reduces the probability that an open chromatin domain (or active transcriptional complex) may form or be maintained in the face of repressive influences of neighboring chromatin.