## Description

These tracks display a synthesis of evidence from different assays as part of the four Open Chromatin track sets. This track displays open chromatin regions and/or transcription factor binding sites identified in multiple cell types by one or more complementary methodologies, DNaseI hypersensitivity (HS) (Duke DNaseI HS), Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) (UNC FAIRE), and chromatin immunoprecipitation (ChIP) for select regulatory factors (UTA TFBS). Each methodology was performed on the same cell type using identical growth conditions. (Note: Data for some or all ChIP experiments may not be available for all cell types). Regions that overlap between methodologies identify regulatory elements that are cross-validated indicating high confidence regions. In addition, multiple lines of evidence suggest that regions detected by a single assay (e.g., DNase-only or FAIRE-only) are also biologically relevant (Song et al., submitted).

DNaseI HS data: DNaseI is an enzyme that has long been used to map general chromatin accessibility, and DNaseI "hypersensitivity" is a feature of active cis-regulatory sequences.The use of this method has led to the discovery of functional regulatory elements that include promoters, enhancers, silencers, insulators, locus control regions, and novel elements. DNaseI hypersensitivity signifies chromatin accessibility following binding of trans-acting factors in place of a canonical nucleosome.

FAIRE data: FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements) is a method to isolate and identify nucleosome-depleted regions of the genome. FAIRE was initially discovered in yeast and subsequently shown to identify active regulatory elements in human cells (Giresi et al., 2007). Similar to DNaseI HS, FAIRE appears to identify functional regulatory elements that include promoters, enhancers, silencers, insulators, locus control regions and novel elements.

ChIP data: ChIP (Chromatin Immunoprecipitation) is a method to identify the specific location of proteins that are directly or indirectly bound to genomic DNA. By identifying the binding location of sequence-specific transcription factors, general transcription machinery components, and chromatin factors, ChIP can help in the functional annotation of the open chromatin regions identified by DNaseI HS mapping and FAIRE.

Input data: As a background control experiment, we sequenced the input genomic DNA sample that was used for ChIP. Crosslinked chromatin is sheared and the crosslinks are reversed without carrying out the immunoprecipitation step. This sample is otherwise processed in a manner identical to the ChIP sample as described below. The input track is useful in revealing potential artifacts arising from the sequence alignment process such as copy number differences between the reference genome and the sequenced samples, as well as regions of poor sequence alignability.

## Display Conventions and Configuration

This track contains multiple subtracks representing different cell types that display individually on the browser. Instructions for configuring tracks with multiple subtracks are here.

To facilitate analyses, each region has been assigned an Open Chromatin Code (OC Code), based on the assay(s) by which it was detected, and a color, based on its level of validation (which was determined by the combination of its OC Code and its statistical significance):

Validated, OC Code = 1:
  - Black: Regions identified as peaks by both the DNaseI HS assay and FAIRE assay. Peaks for DNaseI HS have DNase peak calling p-values `< 0.05` (`-log10(p-value) > 1.3`) and peaks for FAIRE have FAIRE peak calling p-values `< 0.1` (`-log10(p-value) > 1.0`).
Open Chromatin, OC Code = 2 or 3:
  - Blue (high significance): Regions not identified as peaks in both DNaseI HS and FAIRE, but for which the combination of peak calling p-values from these assays using Fisher's combined probability test results in a p-value `< 0.01` (`-log10(p-value) > 2`).
DNase, OC Code = 2:
  - Green (low significance): Regions identified by DNaseI HS as peaks (DNase peak calling p-value `< 0.05` (`-log10(p-value) > 1.3`)) and not identified by FAIRE as peaks (FAIRE peak calling p-value `< 0.1` (`-log10(p-value) > 1.0`)), and with a Fisher's combined DNaseI HS and FAIRE p-value `>= 0.01` (`-log10(p-value) <= 2`).
  - Blue (high significance): see Open Chromatin above.
FAIRE, OC Code = 3:
  - Dark Red (low significance): Regions identified by FAIRE as peaks (FAIRE peak calling p-value `< 0.1` (`-log10(p-value) > 1.0`)) identified by DNaseI HS as peaks (DNase peak calling p-value `< 0.05` (`-log10(p-value) > 1.3`)), and with a Fisher's combined DNaseI HS and FAIRE p-value `>= 0.01` (`-log10(p-value) <= 2`).
  - Blue (high significance): see Open Chromatin above.
ChIP-seq, OC Code = 4:
  - Pink: Regions identified by ChIP-seq as peaks (at least one of the peak calling p-values for the three ChIP experiment is `< 0.05` (`-log10(p-value) > 1.3`)) indicating binding sites for one or more of RNA Pol II, CTCF, and c-Myc described here and not identified by DNaseI HS or FAIRE as peaks. Peaks for ChIP-seq have p-values `< 0.05` (`-log10(p-value) > 1.3`). For RNA Pol II, only sites that overlap annotated transcription start sites by the UCSC Genes track are considered.

All signal values, `-log10`(p-values), and the OC Code is displayed on the detail page for each element and is available in the corresponding bed file.

Metadata for a particular subtrack can be found by clicking the down arrow in the list of subtracks.

## Methods

For each site, the maximum F-Seq Density Signal value has been calculated for each assay that was performed in that cell type. F-Seq employs Parzen kernel density estimation to create base pair scores (Boyle et al., 2008b). Significant regions, or peaks, were determined by fitting the data to a gamma distribution to calculate p-values. Contiguous regions where p-values were below a 0.05 (DNaseI HS, ChIP) or 0.1 (FAIRE) threshold were considered significant. See assay specific description pages ( Duke DNaseI HS, UNC FAIRE and UTA TFBS) for more details.

A Fisher's Combined P-value for DNaseI HS and FAIRE was calculated using Fisher's combined probability test. First, a test statistic is calculated using the formula:

        X2 = -2∑loge(pi)

where `pi` are the p-values calculated for DNaseI HS and FAIRE. `X2` follows a chi-squared distribution, thus a combined p-value can be assigned to this test statistic.

## Release Notes

This is release 1 (May 2011) of this track and is based upon the first release on GRCh37/hg19 of the three Open Chromatin tracks: Duke DNaseI HS, UNC FAIRE, and UTA TFBS.

Enhancer and Insulator Functional assays: A subset of DNase and FAIRE regions were cloned into functional tissue culture reporter assays to test for enhancer and insulator activity. Coordinates and results from these experiments can be found here.

## Credits

These data and annotations were created by a collaboration of multiple institutions (contact: Terry Furey):

- Duke University's Institute for Genome Sciences & Policy (IGSP): Zhancheng Zhang, Lingyun Song, Terry Furey, and Greg Crawford

- University of North Carolina at Chapel Hill: Paul Giresi and Jason Lieb

- Universty of Texas at Austin: Zheng Liu, Ryan McDaniell, Bum-Kyu Lee, and Vishy Iyer

- European Bioinformatics Insitute: Paul Flicek, Damian Keefe, and Ewan Birney

- University of Cambridge, Department of Oncology and CR-UK Cambridge Research Institute (CRI) : Stefan Graf

We thank NHGRI for ENCODE funding support.

## References

Bhinge AA, Kim J, Euskirchen GM, Snyder M, Iyer, VR. Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE). Genome Res. 2007 Jun;17(6):910-6.

Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE. High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008 Jan 25;132(2):311-22.

Boyle AP, Guinney J, Crawford GE, and Furey TS. F-Seq: a feature density estimator for high-throughput sequence tags. Bioinformatics. 2008 Nov 1;24(21):2537-8.

Buck MJ, Nobel AB, Lieb JD. ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data. Genome Biol. 2005;6(11):R97.

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. Nat Methods. 2006 Jul;3(7):503-9.

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.

The ENCODE Project Consortium. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007 Jun 14;447(7146):799-816.

Giresi PG, Kim J, McDaniell RM, Iyer VR, Lieb JD. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolated active regulatory elements in human chromatin. Genome Res. 2007 Jun;17(6):877-85.

Giresi PG, Lieb JD. Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). Methods. 2009 Jul;48(3):233-9.

Li H, Ruan J, and Durbin R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 2008 Nov;18(11):1851-8.

Song L and Crawfor GE. DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb. Protoc.; 2010;Issue 2.

## Data Release Policy

Data users may freely use ENCODE data, but may not, without prior consent, submit publications that use an unpublished ENCODE dataset until nine months following the release of the dataset. This date is listed in the Restricted Until column on the track configuration page and the download page. The full data release policy for ENCODE is available here.