This track displays DNaseI-hypersensitive sites identified using two methods (Dnase-chip and MPSS sequencing) in the GM06990 lymphoblastoid cell line and in non-activated and activated CD4+ T-cells. DNaseI-hypersensitive sites are associated with all types of gene regulatory regions, including promoters, enhancers, silencers, and locus control regions. CD4+ T-cells, also known as helper or inducer T cells, are involved in generating an immune response. CD4+ T-cells are also one of the primary targets of the HIV virus.
Within the subtracks, the gray and black blocks (which appear as vertical lines when the display is zoomed-out) represent probable hypersensitive sites. The darker the blocks, the more likely the site is to be hypersensitive.
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DNaseI hypersensitive sites were isolated using a method called DNase-chip (Crawford et al., unpublished). 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. Each DNase-chip region has a unique identifier, visible when the track is displayed in full or packed mode. The first part of the identifier describes the cell type from which the data was derived. The last number of each identifier indicates the percent of regions that represent valid DNase hypersensitive sites.
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. Each cluster has a unique identifier, visible when the track is displayed in full or packed mode. 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.
Real-time PCR assay was used to validate a randomly selected subset of DNase-chip regions. Approximately 20-30% of regions detected in only a single DNase concentration are valid. These clusters are shown in light gray. 50-80% of regions detected in 2 out of 3 DNase concentrations are valid, indicated by dark gray. 90% of regions detected in all 3 DNase concentrations are valid, shown in black.
This data set includes elements for all 44 ENCODE regions.
Real-time PCR assay 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.
These data were produced at the Collins Lab at NHGRI. Thanks to Gregory E. Crawford and Francis S. Collins for supplying the information for this track.
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):597-8.
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.)
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.