Rapid genome-scale mapping of chromatin accessibility in tissue.
BACKGROUND
The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples.


RESULTS
Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh.


CONCLUSIONS
The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance.
Laboratory of Receptor Biology and Gene Expression, 41 Library Dr. National Cancer Institute, Building 41 B602, Bethesda, MD, 20892, USA.
Lars
L
Grøntved
Gene Regulation Section Laboratory of Pathology, National Cancer Institute, NIH, Building 10, Room 2 N106, Bethesda, MD, 20892, USA.
Russell
R
Bandle
Laboratory of Receptor Biology and Gene Expression, 41 Library Dr. National Cancer Institute, Building 41 B602, Bethesda, MD, 20892, USA.
Sam
S
John
Laboratory of Receptor Biology and Gene Expression, 41 Library Dr. National Cancer Institute, Building 41 B602, Bethesda, MD, 20892, USA.
Songjoon
S
Baek
Gene Regulation Section Laboratory of Pathology, National Cancer Institute, NIH, Building 10, Room 2 N106, Bethesda, MD, 20892, USA.
Hye-Jung
HJ
Chung
Section on Endocrine Physiology, National Institute of Child Health and Development, NIH, Building 10, Room 1-3330, Bethesda, MD, 20892, USA.
Ying
Y
Liu
Section on Endocrine Physiology, National Institute of Child Health and Development, NIH, Building 10, Room 1-3330, Bethesda, MD, 20892, USA.
Greti
G
Aguilera
Laboratory of Pathology, National Cancer Institute, NIH, Building 10, Room 2 N212, Bethesda, MD, 20892, USA.
Carl
C
Oberholtzer
Laboratory of Receptor Biology and Gene Expression, 41 Library Dr. National Cancer Institute, Building 41 B602, Bethesda, MD, 20892, USA.
Gordon L
GL
Hager
Gene Regulation Section Laboratory of Pathology, National Cancer Institute, NIH, Building 10, Room 2 N106, Bethesda, MD, 20892, USA.
David
D
Levens