## 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). In living cells, functionally active cis-regulatory elements bear a unifying feature, which is a chromatin-based epigenetic signature known as nuclease hypersensitivity (Elgin, 1988; Gross and Garrard, 1988; Wolffe, 1998). This track presents the results of a collaboration between J. Craig Venter Institute (JCVI, Rockville MD) and the European Institute of Oncology (Milan, Italy) to isolate nuclease accessible sites (NAS) from primary human CD34+ hematopoietic stem and progenitor cells, and from CD34- cells, maturating myeloid cells generated by in vitro differentiation of CD34+ cells (Gargiulo et al., submitted). This effort made use of a method (originally developed at Sangamo BioSciences, Richmond, CA) to isolate such NAS from living cells using restriction enzymes (RE), leading to minimal, if any, contamination from bulk DNA. High throughput 454 sequencing was then used to generate NAS libraries in CD34+ and CD34- cells: this technology has been named "NA-Seq" (Gargiulo et al., submitted).

## Display Conventions

The track annotates the location of NAS in the genome of human CD34+ and CD34- cells in the form of tags, generated by NA-Seq and obtained by merging NAS within 600 bp. Note that the method identifies a specific position in chromatin that is sensitive to nucleases, but does not map the boundaries of a regulatory element per se. A conservative estimate of element size would be the space 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; Boyle, 2008).

## Methods

CD34+ cells (enriched in hematopoietic stem and progenitor 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 leukaphereses, 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 NAS using the nuclease hypersensitive site isolation protocol (Gargiulo et al., submitted).

## Verification

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 (Fyodor Urnov, unpublished results). These data confirmed that the method yields very high-content libraries of active cis-regulatory DNA elements, supporting its application to human CD34+ cells. In collaboration with scientists at the J. Craig Venter Institute and the European Institute of Oncology, libraries of NAS were prepared using this method in HT 454 sequencing from CD34+ and CD34- cells, and showed that 41 out of 51 randomly chosen clones - >80% - coincided with DNAse I hypersensitive sites (Gargiulo et al., submitted).

## Credits

The library of Nuclease Accessible sites (NAS) from human CD34+/CD34- cells was prepared and validated by Saverio Minucci and colleagues at the European Institute of Oncology. Sequencing was performed by Sam Levy and colleagues (J. Craig Venter Institute). This method was initially developed and validated by Fyodor Urnov, Alan Wolffe, and colleagues at Sangamo BioSciences, Inc.

## References

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

Gargiulo G, Levy S, et al. A Global Analysis of chromatin Accessibility and Dynamics during Hematopoietic Differentiation. Submitted.

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

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

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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.