Distal Regions of the Human IFNG Locus Direct Cell Type-Specific Expression

Abstract

Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG uses cis-regulatory elements with cell type-restricted function.

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

“…Using RecA mediated homologous recombination, 97 mixed CAA-CAG repeats were introduced into the human htt gene in a BAC to build a mouse model of huntingtin's disease [33]. Transgenic mice carrying human BAC clones with 1 kb deletions made by RedET-recombination were used to functionally identify cell lineage-specific regulatory elements 30 kb upstream of the IFNG gene [34]. Recently a moderate throughput BAC modification procedure has been described that uses RedET recombination to introduce the GFP cassette and iTol2 for integration into zebrafish chromosomes [35].…”

“…It has the advantage of using shorter length homologous sequences for recombination to introduce exogenous DNA cassettes into BACs [23]. Both methods have been widely used to engineer a variety of alterations in BAC DNA, such as introducing reporter gene cassettes in frame into the first exon of the target gene, mutating sequences at a target site, and introducing loxP sites [24][25][26][27][28][29][30][31][32][33][34]. Thus BACs of ~200 kb size and containing the β-globin gene were functionalized with EGFP reporter gene using RedET-recombination and expressed in stable erythropoietic cell lines [29].…”

“…Because all members of the deletion library contain identical changes at the loxP or lox511 end, comparisons of expression patterns between any two enhancer-trap BACs from the same deletion series are more meaningful as the modifications in each remain constant. Thus BACs can be "scanned" with enhancer-traps to identify distal regulatory elements; and this approach is likely to be less biased than other methods where mutations are targeted to specific sites in the BAC because there is no need to select sequences to test their regulatory potential [30][31][32]34]. In situations where regulatory function is conserved without sequence similarity [10][11][12][13][14], choosing the correct sequence to test might be very difficult as many of the players involved in fine tuning the regulation of gene expression remain unknown.…”

Trapping Enhancers by Transgenic Expression of BACs Engineered in Bacteria with loxP Transposons

“…Using RecA mediated homologous recombination, 97 mixed CAA-CAG repeats were introduced into the human htt gene in a BAC to build a mouse model of huntingtin's disease [51]. Transgenic mice carrying human BAC clones with 1 kb deletions made by RedET-recombination were used to functionally identify cell lineage-specific regulatory elements 30 kb upstream of the IFNG gene [52]. Recently a moderate throughput BAC modification procedure has been described that uses RedET recombination to introduce the GFP cassette and iTol2 for integration into zebrafish chromosomes [53].…”

“…It has the advantage of using shorter homologous sequences for recombination to introduce exogenous DNA cassettes into BACs [41]. Both methods have been widely used to engineer a variety of alterations in BAC DNA, such as introducing reporter gene cassettes in frame into the first exon of the target gene, mutating sequences at a chosen site, and introducing loxP sequences [42][43][44][45][46][47][48][49][50][51][52]. Thus BACs of ~200 kb size and containing the β-globin gene were functionalized with EGFP reporter gene using RedET-recombination and expressed in stable erythropoietic cell lines [47].…”