Insulator Elements in Erythrocyte Membrane Genes

Insulators are DNA sequences and associated binding proteins that establish and/or maintain the boundaries between euchromatin and heterochromatin. One type of insulator establishes chromatin domains to separate enhancers and promoters and prevent their interaction (enhancer-blocking insulators) whereas another type creates a barrier to protect against heterochromatin-mediated gene silencing (barrier insulators). In the well-characterized chicken beta-globin LCR 5′HS4 insulator, binding of CTCF mediates enhancer-blocking functions and USF proteins mediate barrier activity. Because varying transcripts of erythrocyte membrane protein genes are expressed in both erythroid and nonerythroid cells, we hypothesized that that insulator elements that bind USF and CTCF participate in their regulation. Advances in technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNA-associated proteins on a genome-wide scale. Coupling chromatin immunoprecipitation to microarrays that contain genomic regions (ChIP-chip) is a high resolution technique available for mapping protein-DNA interactions in vivo. We used ChIP-chip to identify CTCF and USF factor binding sites with potential insulator function that regulate membrane protein gene expression in erythroid cells. ChIP was performed with K562 and HeLa cells using antibodies against CTCF, USF1, and USF2. DNA obtained from these IPs was hybridized to a custom NimbleGen high-density human genomic DNA microarray. Chip probes were ~50bp in length, Tm ≥76°C, tiled ~65bp apart. Regions of repetitive DNA excluded. The chip included 15 erythrocyte membrane protein genes, most encoding complex loci with multiple tissue-, cell-, and developmental stage-specific transcripts, including alpha spectrin, beta spectrin, ankyrin, spectrin, band 3, ƒnalpha adducin, beta adducin, gamma adducin, ICAM4, erythroid associated membrane protein, protein 4.1R, protein 4.2, dematin, beta actin, tropomodulin, and tropomyosin. Each gene plus 50 to 100kb of flanking DNA were included on the chip. Binding sites on the custom DNA array were identified using the Tamalpais peak calling algorithm using L1–L3 level of stringency (