An Active Chromatin Hub Involving the Three Alternate Promoter/First Exons of the Ankyrin-1 (ANK-1) Gene

Abstract 3862

The Ankyrin-1 (ANK-1) gene is an example of the many mammalian genes with alternate promoters in which transcripts containing distinct first exons are fused to a common exon 2 in different cell types. The ANK-1E promoter/first exon is active in only erythroid cells and is located between a promoter/first exon located 40 kb upstream that is expressed exclusively in brain and muscle cells (ANK-1B) and a promoter/first exon located 16 kb downstream that is expressed in many cell types (ANK-1A). We have previously shown that the ANK-1E promoter/first exon is flanked by two DNase I hypersensitive sites (HS). The ANK-1E 5′HS is located in the promoter region, while two adjacent HS (3′HS 1&2) are located ∼6 kb downstream. We hypothesize that the erythroid-specific activation of the ANK-1E promoter/first exon involves both changes in transcription factor occupancy and in chromatin structure. Using whole-genome Chromatin Immune Precipitation (ChIPSeq) and whole-genome mRNA analysis (RNASeq), we have made a detailed study of Erythroid Krupple-like Factor (EKLF) occupancy and mRNA levels in sorted mouse erythroid progenitor cells and definitive erythroblasts. In erythroid progenitor cells, EKLF occupies the Ank-1E 5′HS, and the level of Ank-1E mRNA is relatively low. In erythroblasts, EKLF is not detected at either Ank-1E 5′HS or the Ank-1E 3′HS and the Ank-1 mRNA level is increased more than 2-fold, consistent with EKLF acting as a transcriptional repressor. We have also shown that the ANK-1E 5′HS and the 3′HS region are both occupied by the transcription factors GATA-1 and NF-E2, although EMSA assays have shown that NF-E2 does not bind to the 5′HS region, and that GATA-1 does not bind to the 3′HS region in vitro. Addition of the NF-E2 site in 3′HS1 to the ANK-1E promoter increases expression 4-fold indicating a potential interaction of these two regions. We have recently demonstrated that both the ANK-1E 5′HS and 3′HS2 act as barrier elements that prevent gene silencing in cultured erythroid cells and transgenic mice. Consistent with the functional observations, in erythroid cells both ANK-1E 5′HS and the 3′HS region are occupied by the barrier-associated proteins USF-1 and -2, PRMT-1 and -4, CTCF and SRC1, while the ANK-1A region is occupied by the barrier-associated proteins USF-2, PRMT-1 and -4, but not USF-1 and CTCF. We hypothesized that the HS flanking the ANK-1E promoter/first exon form a chromatin loop that brings both the transcription factors and barrier proteins into proximity. Using the chromatin conformation capture assay we demonstrated an interaction between the Ank-1E 5′HS and the 3′HS region. Analysis of the three dimensional structure of the 100 kb region upstream of the mouse Ank-1 exon 2 region was performed using the chromatin conformation capture carbon copy (5C) assay. We designed a set of 12 forward primers on the three Ank-1 promoter/first exons and the HS that flank them, and a set of 264 reverse primers to analyze all interactions in this region. In mouse fetal liver erythroid cells, the strongest interaction is between Ank-1E 5′HS and a region 15 kb 3′ to the Ank-1B region. In addition, the Ank-1B promoter/first exon region interacts with both the Ank-1A region and a region 30 kb downstream of the Ank-1B region forming a ‘rosette’ that includes Ank-1E. We propose a model in which the formation of an active chromatin hub in erythroid cells which is facilitated by erythroid transcription factors and stabilized by the barrier-associated proteins.