## Description

This track displays DNaseI-hypersensitive sites identified using two methods (DNase-chip and MPSS sequencing) in seven human cell types:

- primary unactivated and activated CD4+ T cells
- GM06990 lymphoblastoid
- HeLa S3 cervical carcinoma (Puck et al., 1956)
- HepG2 liver carcinoma
- H9 human undifferentiated embryonic stem (ES) (Thomson et al., 1998)
- IMR90 human fibroblast
- K562 myeloid leukemia-derived (Klein et al., 1976)

DNaseI-hypersensitive sites are associated with all types of gene regulatory regions, including promoters, enhancers, silencers, insulators, and locus control regions.

## Display Conventions and Configuration

The subtracks within this track are grouped into three sections:

- Raw subtracks display log2 ratio data averaged from three biological replicates and three DNase concentrations.
- Pval subtracks show significant regions that likely represent valid DNaseI-hypersensitive sites based on the raw data. The higher the score for the region, the more likely the site is to be hypersensitive. Regions have unique identifiers that are prefixed with the cell type. For display purposes, the p value scores were mapped to integer scores in the range 0-1000. Regions are displayed in a range of light gray to black, based on score.
- MPSS subtracks show hypersensitive sites determined by massively parallel signature sequencing (MPSS). Each cluster has a unique identifier. The last digit of each identifier represents the number of sequences that map within that particular cluster. The sequence number is also reflected in the score, e.g. a cluster of two sequences scores 500, three sequences scores 750 and four or more sequences scores 1000. Sites are displayed in a range of light gray to black, based on score.

The "Raw" and "Pval" subtracks are displayed by default. Use the checkboxes on the Track Settings page to change the subtracks displayed.

## Methods

### DNase-Chip

DNaseI hypersensitive sites were isolated using a method called DNase-chip (Crawford et al., 2006). Briefly, DNaseI digested ends from intact chromatin were captured using three different DNase concentrations as well as three biological replicates. This material was amplified, labeled, and hybridized to NimbleGen ENCODE tiled microarrays.

H9 human ES cells (Thomson et al., 1998) were cultured on a feeder layer of mitotically inactivated mouse embryo fibroblasts. For analysis, human ES cell colonies were separated away from the feeder layer and processed for DNaseI hypersensitive site mapping. Cultures were routinely inspected by immunohistochemistry, flow cytometry, and microarray to ensure that the human ES cells were in the undifferentiated state.

For the DNase-chip experiments, the raw data were averaged from nine hybridizations per cell type. The Pval scores represent -log10 p values as determined by the ACME (Algorithm for Capturing Microarray Enrichment) program (Scacheri et al., 2006). Only regions that had p value < 0.001 were included. For display in the Genome Browser, the p value scores were mapped to integer scores in the range 0-1000 using the following formula: score = (pVal * 35) + 100. The -log10 p values can be viewed using the Table Browser.

### MPSS Sequencing

Primary human CD4+ T cells were activated by incubation with anti-CD3 and anti-CD28 antibodies for 24 hours. DNaseI-hypersensitive sites were cloned from the cells before and after activation, and sequenced using massively parallel signature sequencing (Brenner et al., 2000; Crawford et al., 2006). Only those clusters of multiple DNaseI library sequences that map within 500 bases of each other are displayed.

## Verification

### DNase-Chip

A real-time PCR assay (McArthur et al., 2001; Crawford et al. , 2004) was used to validate a randomly selected subset of DNase-chip regions. For the New DNase-chip, the Sensitivity of DNase-chip was determined to be > 86% and Specificity to be > 97%. Approximately 20-30% of regions detected in only a single DNase concentration are valid. 50-80% of regions detected in two out of three DNase concentrations are valid (the exact percentage depends on which two DNase concentrations had significant signal). 90% of regions detected in all three DNase concentrations are valid.

This data set includes elements for all 44 ENCODE regions.

### MPSS Sequencing

Real-time PCR was used to verify valid DNaseI-hypersensitive sites. Approximately 50% of clusters of two sequences are valid. These clusters are shown in light gray. 80% of clusters of three sequences are valid, indicated by dark gray. 100% of clusters of four or more sequences are valid, shown in black.

This data set includes confirmed elements for 35 of the 44 ENCODE regions. It is estimated that these data identify 10-20% of all hypersensitive sites within CD4+ T cells. Further sequencing will be required to identify additional sites. MPSS data from the whole genome can be found in the Expression and Regulation track group (NHGRI DNaseI-HS track).

## Credits

These data were produced at the Crawford Lab at Duke University, and at the Collins Lab at NHGRI. Thanks to Gregory E. Crawford and Francis S. Collins for supplying the information for this track. H9 cells were grown in collaboration with Ron McKay and Paul Tesar at the National Institute of Neurological Disorders and Stroke (NINDS)—an institute of the National Institutes of Health (NIH).

## References

Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M et al. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol. 2000 Jun;18(6):630-4.

Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, Collins FS. DNase-chip: A high resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nature Methods. 2006 Jul;3(7):503-9.

Crawford GE, Holt IE, Mullikin JC, Tai D, Blakesley R, Bouffard G, Young A, Masiello C, Green ED, Wolfsberg TG et al. Identifying gene regulatory elements by genome-wide recovery of DNase hypersensitive sites. Proc Natl Acad Sci USA. 2004 Jan 27;101(4):992-7.

Crawford GE, Holt IE, Whittle J, Webb BD, Tai D, Davis S, Margulies EH, Chen Y, Bernat JA, Ginsburg D et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 2006 Jan;16(1):123-31. (See also NHGRI's data site for the project.)

Klein E, Ben-Bassat H, Neumann H, Ralph P, Zeuthen J, Polliack A, Vanky F. Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia. Int J Cancer. 1976 Oct 15;18(4):421-31.

McArthur M, Gerum S, Stamatoyannopoulos G. Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse beta-globin LCR . J Mol Biol. 2001 Oct 12;313(1):27-34.

Puck TT, Marcus PI, Cieciura SJ. Clonal growth of mammalian cells in vitro: growth characteristics of colonies from single HeLa cells with and without a "feeder" layer. J Exp Med. 1956 Feb 1;103(2):273-83.

Scacheri PC, Crawford GE, Davis S. Statistics for ChIP-chip and DNase hypersensitivity experiments on NimbleGen arrays. Methods Enzymol. 2006;411:270-82.

Thomson JA, Itskovitz-Eldor J, Shapirom SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7.