Cis and Trans Regulation of the ANK1 Locus
Yocum, Ashley Owen
Many mammalian loci have alternate promoters in which transcripts contain distinct first exons that are fused to a common exon 2 in different cell types. The human ankyrin 1 gene (ANK1) is a classic example of this type of locus. ANK1 contains three tissue specific alternative promoters/first exons. ANK1B is expressed exclusively in brain and muscle cells and is 138 kb upstream of exon 2. ANK1E is active only in erythroid cells and is 39 kb upstream of exon 2. ANK1A is expressed in many cell types and is 9 kb upstream of exon 2. We have previously shown that the ANK1E core promoter contains a 5' DNase I hypersensitive site (5'HS) with barrier insulator activity that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and subsequently Hereditary Spherocytosis. Here we demonstrate a second ANK1E barrier insulator located within an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter, which defines the 3' boundary of an erythroid specific active chromatin domain. We show that ANK1E 5'HS and 3'HS region prevent gene silencing in cultured erythroid cells and transgenic mice. In erythroid cells both the ANK1E 5'HS and the 3'HS regions are occupied by the barrier-associated proteins USF-1 and -2, PRMT-1 and -4, and CTCF which is consistent with our functional observations. In addition both the 5'HS and 3'HS regions are occupied by the transcription factors GATA1 and NF-E2. Addition of the NF-E2 site in the 3'HS to the ANK1E promoter increases expression 4-fold, indicating a potential interaction of these two regions. We show that in erythroid cells, a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including ANK1E. This is the first demonstration of a role for barrier insulators in determining which alternative promoter is active in a specific cell type, which may be applicable to the ~35% of mammalian genes with alternative promoters with distinct expression patterns.
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