Defining the Active Chromatin Domain of the Erythroid Ankyrin Promoter

• Published in: Blood 2004-11, Vol.104 (11), p.575-575
• Main Authors: Liem, Robert I., Cline, Amanda P., Garrett-Beal, Lisa J., Owen, Ashley N., Gallagher, Patrick G., Bodine, David M.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V104.11.575.575

Erythrocyte ankyrin forms the bridge between the spectrin/actin cytoskeleton and the red cell membrane protein band 3. Mutations resulting in defective or deficient ankyrin are the most common cause of Hereditary Spherocytosis. The erythroid ankyrin promoter (ankR) lies between two other ankyrin promoters, one that is active in neuronal cells and the other in many cell types. Active genes are generally found in DNase I sensitive regions of chromatin while regulatory sequences (promoters, enhancers, boundary elements) are associated with DNase I Hypersensitive Sites (HS). We hypothesized that the chromatin surrounding ankR determines its activity. In human K562 cells and mouse fetal liver cells a DNase I HS (5′HS) maps between −300 and −100 of ankR. 5′HS marks the end of a DNase I resistant chromatin extends at least 10 kb upstream, indicating that this chromatin is inactive in erythroid cells. We have previously shown that a 300 bp fragment containing ankR was sufficient to direct erythroid specific, copy number dependent, position independent and uniform expression of a linked γ-globin gene in transgenic mice. Identical results were obtained with mice containing −2.7 kb and −656 bp ankyrin promoters, as expected, and 5′ HS was present in fetal liver chromatin of all 8 lines examined. Downstream of 5′HS is a 5.7 kb DNase I sensitive region that includes a 500 bp enhancer-like element near the 3′ end. At the 3′ end of the DNase I sensitive domain are 2 HS (3′HS) that mark the beginning of a DNase I resistant region that extends at least 3 kb downstream. AnkR is inactive in non-erythroid HeLa, Jurkat and Sh-SY5Y (neuronal) cells. Although both 5′HS and 3′HS are present in these cells, the intervening 5.7 kb region is DNase I resistant indicating that erythroid specific factors are required for the activation of ankR. Boundary elements (insulators) are found between DNase I resistant and DNase I sensitive chromatin and have the ability to prevent the transgene silencing at ectopic sites in the genome. To test whether the 5′HS and 3′HS function as boundary elements, a β-globin promoter/EGFP transgene was flanked by 200 bp fragments containing either 5′HS or 3′HS. While 0/8 K562 cell lines with the transgene alone expressed GFP, GFP expression was observed in 12/12 5′HS and 11/12 3′HS K562 cell lines (χ2=12.0, p