1. ¹Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115
2. ²Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei 112
3. ³Institute of Life Sciences, National Defense University, Nei-hu, Taipei 114
4. ⁴Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Miaoli County 350
5. ⁵Department of Biochemical Engineering, Kao Yuan University, Lu-Chu Hsiang, Kaohsiung County 821

Correspondence: Che-Kun James Shen, Tel: 011-886-2-27824188; Fax: 011-886-2-27884177; E-mail: ckshen@imb.sinica.edu.tw

^*These two authors contributed equally to this work.

Received 6 October 2005; Revised 6 January 2006; Accepted 12 January 2006.

Abstract

EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation (IP) qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation (ChIP) assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells (MEL) in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase II, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE (HS26). This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.