SGMO/EIO CD34 HS Track Settings
 
Sangamo - Eur. Inst. Oncology DNase Hypersensitive Sites   (All Regulation tracks)

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Assembly: Human May 2004 (NCBI35/hg17)
Data last updated at UCSC: 2005-08-15

Description

Genes in metazoa are controlled by a complex array of cis-regulatory elements that include core and distal promoters, enhancers, insulators, silencers etc (Levine and Tjian, 2003). A unifying feature of such elements active in cell nuclei is a chromatin-based epigenetic signature known as a nuclease hypersensitive site (Elgin, 1988; Gross and Garrard, 1988; Wolffe, 1998). This track presents the results of a collaboration between Sangamo BioSciences, Inc. and the European Institute of Oncology to isolate such regulatory elements from human CD34+ hematopoietic stem cells (Urnov et al., submitted). This effort made use of a method developed at Sangamo BioSciences to isolate such nuclease hypersensitive sites from living cells with minimal, if any, contamination from bulk DNA (Urnov et al., submitted; US patent pending).

Display Conventions

The track annotates the location of 3,314 cis-regulatory elements in human CD34+ cells in the human genome in the form of 40 bp tags. Note the method identifies a specific position in chromatin that is hypersensitive to nuclease, but does not map the boundaries of the regulatory element per se. A conservative estimate of element size would be that occupied by one nucleosome, i.e., 180 - 200 bp surrounding the tag, although there is precedent in the literature for nuclease hypersensitive sites that span more than the length of one nucleosome (Turner, 2001; Wolffe, 1998).

Methods

CD34+ cells (enriched in hematopoietic stem cells) were prepared from healthy donors following guidelines established by the Ethics Committee of the European Institute of Oncology (IEO), Milan. Mobilization of CD34+ cells to the peripheral blood was stimulated by G-CSF treatment according to standard procedures. After mobilization, donors were subjected to leukophoresis, and <10% of the sample was used in the experiment. CD34+ cells were purified using a magnetic positive selection procedure ("EASYSEP"; Stemcell, Vancouver, Canada). Purity of separation was evaluated by FACS after staining with an anti-Human CD34 FITC-conjugate antibody (Stemcell). Upon purification, the cell cycle status of the CD34+ cells was monitored by propidium iodide staining and FACS analysis. G0/G1 cells varied from approximately 90% to >95% of the total cells. Cells were immediately used for the isolation of cis-regulatory DNA elements using the nuclease hypersensitive site isolation protocol developed at Sangamo (Urnov et al., submitted).

Verification

In collaboration with scientists at the J. Craig Venter Institute and the European Institute of Oncology, the method was initially validated on human tissue culture cells by examining the colocalization of DNA fragments isolated from cells with experimentally determined nuclease hypersensitive sites in chromatin as mapped by indirect end-labeling and Southern blotting (Nedospasov and Georgiev, 1980; Wu, 1980). Nineteen out of nineteen randomly chosen clones from those libraries represented bona fide DNAse I hypersensitive sites in chromatin (Urnov et al., submitted). These data confirmed that the method yields very high-content libraries of active cis- regulatory DNA elements, supporting its application to human CD34+ cells. Analysis of libraries of cis-regulatory elements prepared using this method from CD34+ cells showed that 50 out of 55 randomly chosen clones — 91% — coincided with DNAse I hypersensitive sites (Urnov et al., submitted).

Credits

The library of regulatory DNA elements from human CD34+ cells was prepared, sequenced, and validated by Saverio Minucci and colleagues at the European Institute of Oncology, using a method developed by Fyodor Urnov, Alan Wolffe, and colleagues at Sangamo BioSciences, and validated in collaboration with Sam Levy and colleagues (J. Craig Venter Institute).

References

Elgin SC. The formation and function of DNase I hypersensitive sites in the process of gene activation. J Biol Chem. 1988 Dec 25;263(36):19259-62.

Gross DS, Garrard WT. Nuclease hypersensitive sites in chromatin. Ann Rev Biochem. 1988;57:159-197.

Levine M, Tjian R. Transcription regulation and animal diversity. Nature. 2003 Jul 10;424(6945):147-51.

Nedospasov S, Georgiev G. Non-random cleavage of SV40 DNA in the compact minichromosome and free in solution by micrococcal nuclease. Biochem Biophys Res Commun. 1980 Jan 29;92(2):532-9.

Turner BM. Chromatin and Gene Regulation: Mechanisms in Epigenetics. Blackwell Publishers, Oxford. 2001.

Urnov FD, Minucci S, Levy S et al. Genome-wide chromatin-based isolation of active cis-regulatory DNA elements from human cells. Submitted.

Wolffe AP. Chromatin Structure and Function. Academic Press, San Diego, CA. 1998.

Wu C. The 5' ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature. 1980 Aug 28;286(5776):854-60.