Rapid genome-scale mapping of chromatin accessibility in tissue
- Lars Grøntved†1,
- Russell Bandle†2,
- Sam John†1,
- Songjoon Baek1,
- Hye-Jung Chung2,
- Ying Liu3,
- Greti Aguilera3,
- Carl Oberholtzer4Email author,
- Gordon L Hager1Email author and
- David Levens2Email author
© Grøntved et al.; licensee BioMed Central Ltd. 2012
Received: 14 February 2012
Accepted: 18 May 2012
Published: 26 June 2012
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.
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.
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.
KeywordsChromatin accessibility Tissue TACh Benzonase Cyanase DNase I
The coupling of next-generation sequencing methodologies with classical enzymatic chromatin digestion approaches (eg. DNase I and MNase) has provided global, high-resolution information on chromatin features such as nucleosome positioning and chromatin accessibility [1, 2]. DNase-Seq has emerged as a powerful genome-wide tool to identify and characterize chromatin transitions (DNase I hypersensitive sites or DHS) in regulatory regions across a range of biological processes and cell lines [1, 3–5]. Current methodologies to generate genome-wide DHS data start with large numbers of fresh cells from which sizeable quantities of high quality nuclei need to be generated . This has restricted application of the DNase I approach to cells in culture and freshly isolated tissue and has prevented the use of this technology on clinical tissue samples that are typically scarce, stored frozen and from which nuclei are difficult to obtain.
The use of DNase I as a probe of chromatin accessibility was initially described three decades ago [7, 8], where partial digestion of chromatin with DNase I indentified DHS in promoters of heat shock genes. The nucleosomal steric hindrance of DNase I access to DNA is similar to that of transcription factors (TF) and the degree of DNase I accessibility correlates with the level of TF occupancy . Thus DNase I hypersensitivity is considered an independent, unbiased probe for TF accessibility. Today, the primary commercial sources of DNase I are either recombinant proteins or purified enzymes from bovine pancreas. However, DNase I is known to have drawbacks that restrict its broad-spectrum use. Specifically, it is known to have a narrow effective concentration window, requiring fastidious titration of enzyme and chromatin to obtain optimal partial digestion [6, 7]. Moreover, DNase I is inhibited by high levels of actin  and because the actin content varies amongst cell and tissue types, each biological sample must be individually titrated. Preparation of nuclei prior to digestion reduces actin contamination, while in situ NP40 lysis leaving adherent nuclei also allows DNase I treatment of cells grown as a monolayer .
In order to define accessible chromatin compartments in samples derived from frozen tissue, where cell numbers are unknown and nuclear preparation is problematic, we hypothesized that digestion of chromatin with more robust nucleases that function in a coarse environment as well as, over broad concentration ranges, might enable the determination of chromatin accessibility in tissue samples. Benzonase, a recombinant endonuclease derived from Serratia marcescens, is composed of two identical subunits of 30 kDa, requires divalent cations for full activity and is generally used to clear cellular protein extracts of DNA and RNA prior to downstream analysis . It digests DNA and RNA efficiently under a range of conditions to nucleotides of 2–5 base pairs. Although the enzyme is able to cleave DNA at all positions, it has been reported to have a relative preference for GC rich regions over dA/dT tracts . Cyanase is a less described non-Serratia recombinant endonuclease that, like Benzonase, efficiently digests DNA and RNA under harsh conditions. Here we introduce a novel technique that combines rapid processing of frozen tissue using Benzonase and Cyanase to specifically identify Tissue Accessible Chromatin (TACh) from frozen specimens. Accessible regions identified with TACh correlate with features of euchromatin and levels of transcription, suggesting that these accessible regions are indeed regulatory. We propose that TACh will be a valuable tool to identify the physiological or pathological regulatory features of chromatin from clinical materials.
Benzonase and Cyanase as probes for chromatin accessibility
To set a standard for the fidelity of Benzonase and Cyanase as a probe for chromatin accessibility, we initially performed a conventional nuclease hypersensitivity assay using cultured cells. Human promyelocytic leukemia cells (HL-60) grown in suspension were isolated, resuspended hypotonic buffer and incubated with increasing concentrations of Benzonase and Cyanase. Accessible regions at the c-myc promoter were compared using indirect-end labeling and Southern blotting as previously described . We show that Benzonase and Cyanase yielded the same pattern of hypersensitive regions expected for DNase I , demonstrating that Benzonase and Cyanase are useful probes for chromatin accessibility (Figure 1B).
Identification of accessible chromatin in frozen tissue
Correlation of Benzonase hotspots with euchromatin and TSS of active genes
Benzonase and Cyanase accessible regions overlap with DNase I hotspots
Sequence bias for endonucleases
Nuclease accessible sites are maintained by the targeted recruitment of remodeling complexes by transcription factors . The transcription factor FOXA2 has been shown to bind AT rich DNA motifs , whereas SREBP has been suggested to bind regions together with SP1  with a resulting preference for GC-rich motifs embedded in CpG islands. Accordingly, the tags sequenced following DNase I digestion were recovered at higher rates from FOXA2 binding sites compared to the tags sequenced using Benzonase-Cyanase digestion (Figure 6F). In contrast, tags from Benzonase-Cyanase digestion were recovered more efficiently at SREBP binding sites (Figure 6G), emphasizing that the differences in base-selectivity at cleavage sites for these enzymes translates into differentially efficient capacities to interrogate the compartments associated with different transcription factors. Though largely similar, Benzonase-Cyanase and DNase I possess unique features that widen the capacity for genome-wide interrogation of chromatin accessibility.
Identification of nuclease accessible sites is a powerful approach to annotate regulatory regions of the genome. In cell lines and fresh tissue where nuclei can be isolated with high efficiency, DNase I has been used for decades to probe for accessibility. However DNase I is inhibited by actin and DNase I can therefore not be used on frozen tissue samples where nuclei cannot be purified in sufficient amounts while maintaining nuclear integrity. Here, we show the use of two other nucleases, Benzonase and Cyanase, in probing chromatin accessibility in frozen whole tissue samples. The digestion patterns of chromatin with Benzonase and Cyanase are remarkably similar and the identified accessible regions correlate with low nucleosomal occupancy, epigenetic marks of euchromatin and levels of transcription of proximal genes, validating the use of TACh for identification of regulatory elements in the genome. Comparisons with DNase I accessibility in liver tissue display a significant overlap between DNase I accessible sites and accessible regions identified by TACh. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergent between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases, with Benzonase and Cyanase preferring GC-rich sequences and disfavoring AT-rich sequences.
Currently, formaldehyde-assisted isolation of regulatory elements (FAIRE) is the only reported method to identify regulatory elements in frozen tissue samples ; however, FAIRE is an ambiguous approach that relies on the density of proteins at regions of crosslinked chromatin, where less dense regions are identified by FAIRE . As a consequence, FAIRE signals depend greatly on crosslink efficiencies of the tissue material, sonication efficiency and organic extraction steps. FAIRE shows some degree of overlap with DNase I accessible regions, however, certain regions are FAIRE specific and many regions are not identified by FAIRE . Since the TACh procedure like the DNase I assay relies on nuclease accessibility of DNA within chromatin, TACh is a more reliable procedure that is less dependent on extrinsic variables.
For clinical samples, TACh provides several advantages compared to current nuclease based methods. 1) TACh uses frozen tissues, which eliminates the need for immediate processing of freshly acquired tissue and is, therefore, compatible with the acquisition of samples under routine clinical conditions. 2) The ability to store whole tissue, fragments or pulverized powders provides flexibility and the powder ensures easy generation of matched aliquots that can be used for additional genomic experiments. 3) The ability to interrupt the procedure at multiple steps makes the whole process more amenable to clinical situations and laboratories. 4) The quick processing and the use of whole cells are likely to minimize nuclear damage and the loss of dissociated chromatin components. 5) Because the chromatin structure is preserved, it is likely that the same pulverized tissue can serve as the starting material for ChIP-Seq interrogations of the same tissue.
Thus TACh will make it possible to mine the accessible genomes from banks of frozen specimens, including biopsies and resected material from diseased and healthy human tissue, and is likely to assist in understanding the pathophysiology of a number of disease states. In particular, since TACh efficiently identifies accessible CpG islands, the method may be used to characterize chromatin structure rearrangements during progression of cancer, which are often associated with abnormal DNA methylation at CpG island rich promoters, leading to deregulation of numerous genes . Identification of genomic regions that specifically change accessibility during tumorigenesis may have significant prognostic value.
The described TACh methodology is a robust method for highly sensitive and comprehensive detection of accessible chromatin in samples derived from frozen tissue. The robustness and quick processing time of the assay provides feasible analysis of multiple tissue biopsies and we propose that application of TACh on clinically derived tissue material will provide knowledge on changes in chromatin accessability during progression of diseases.
HL-60 (ATCC) suspension cells were cultured in RPMI + 10% FBS in CO2 at 37°C.
Male C57BL/6 mice, 8-week old purchased from Harlam Sprague Dawley (Frederick, MD), were maintained according to the NIH guidelines with a 14-h light, 10-h dark cycle and free access to food and water. Mice were killed by decapitation or cervical dislocation and livers were immediately sectioned into 5-10 mm cubes and frozen in liquid nitrogen or processed for nuclei purification. All animal procedures were approved by the Animal Users and Care Committee, NICHD and NCI, NIH.
TACh on whole cells
Cells were collected by centrifugation, washed twice in ice cold cellular wash buffer (CWB) (20 mM TrisHCl pH 7.5, 137 mM NaCl, 1 mM EDTA , 10 mM sodium butyrate, 10 mM sodium orthovanadate, 2 mM sodium fluoride, protease inhibitor cocktial (Roche)) and resuspended in (40 million cells/ml) hypertonic lysis buffer (HLB) (20 mM TrisHCl pH 7.5, 2 mM EDTA, 1 mM EGTA, 0.5% glycerol, 20 mM sodium butyrate, 2 mM sodium orthovanadate, 4 mM sodium fluoride, protease inhibitor cocktial (Roche)). Cells were distributed in 500ul aliquots in 1.5 ml tubes and following addition of 500ul of nuclease digestion (ND) buffer (40 mM TrisHCl pH 8.0, 6 mM MgCl2, 0.3% NP-40, 1% Glycerol) containing a 3-fold dilutions (from 0.125 units/ml to 6U units/ml) of Benzonase (Calbiochem/EMD) or Cyanase (RiboSolutions, Inc). This was mixed gently and incubated for 3 minutes at 37°C. Reactions were terminated by the addition of EDTA (10 mM final) and SDS (0.75% final). Proteinase K was added to a final concentration of 0.5 mg/ml and incubated overnight at 45°C. DNA was purified by Phenol/Chloroform/isoamyl (PCI) extraction, ethanol precipitated and processed for Southern blotting.
Purified nuclease digested DNA was digested with HindIII overnight and then PCI extracted and ethanol precipitated. Precipitated DNA was SacI digested, RNase treated, extracted by PCI and ethanol precipitated. Digested DNA was separated on an agarose gel and transferred to a nylon membrane and incubated with a biotinylated c-myc probe  overnight at 50°C. Membranes were washed, incubated with HRP-conjugated streptavidin solution (kit from KPL, Inc.) and developed using chemiluminescent reagents (ECL Plus from Amersham/GE HealthCare).
TACh on frozen liver tissue
Livers were rapidly removed from mice and frozen immediately in liquid nitrogen. Frozen liver fragments were pulverized using a stainless steel pulverizer (Bessman), pre-chilled in liquid nitrogen. Seven hundred mg of frozen (the procedure may be done on a smaller amount of starting material), pulverized tissue was transferred to a pre-cooled 15 ml tube and suspended in 4 ml HLB (30 mM TrisHCl pH8.0, 2 mM EDTA, 2 mM EGTA, 20 mM sodium butyrate, 2 mM sodium orthovanadate, 4 mM sodium fluoride, protease inhibitor cocktail (Roche)). The suspension was mixed with a 3 cc syringe fitted with a 19 gauge needle and HLB was added to a final volume of 7 ml. The resulting suspension was passed through a 19 gauge needle 5 times, followed by a 22 gauge needle 5 times and finally a 23 gauge 10 times, using a 3 cc syringe. The suspension was distributed in 500ul aliquots in 1.5 ml tubes using a 23 gauge needle and incubated on ice for 5 minutes. 500ul of ND buffer (30 mM TrisHCl pH 8.0, 14 mM MgCl2 0.5% NP-40, 0.2% fatty acid free BSA) containing a 2-fold dilutions of Benzonase (Calbiochem/EMD) or Cyanase (RiboSolutions, Inc) was mixed gently with the 500ul tissue solution and incubated for 3 minutes at 37°C. Reactions were terminated by the addition of a final concentration of 50 mM EDTA and 0.1% SDS. 50ul RNaseA/RNaseT1 (Ambion) was added and the reaction was incubated over night at 40°C. SDS was brought to a final concentration of 0.75% and incubated for 2 hours at 45°C, after which Proteinase K was added (0.8ug/ml final) and incubated overnight at 45°C. DNA fragments of 100-500 bp from a chromatin digestion were purified over sucrose gradients  and precipitated in 0.1 volume NaAc and 0.7 volume isopropanol.
DNase I digestion of chromatin from liver tissue
Livers was removed from mice and homogenized in 8 ml/g tissue of low sucrose buffer (250 mM sucrose, 15 mM Tris–HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM spermidine, protease inhibitors (Roche)) using a Type B dounce. Crude nuclear pellets were washed once in low sucrose buffer and resuspended in 9 volumes of high sucrose buffer (2 M sucrose, 15 mM Tris–HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM spermidine, protease inhibitor cocktail (Roche)). The nuclear suspension was aliquoted in 2.0 ml tubes and centrifuged at 16,000xg for 30 minutes at 4°C. Nuclear pellets were combined in buffer (15 mM Tris–HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM Spermidine, protease inhibitor cocktail (Roche)) and DNase I digestions were performed with 20 million nuclei as previously described (. DNA fragments of 100-500 bp from a chromatin digestion with 60U/ml DNase I (Sigma) were purified using sucrose gradients  and precipitated in 0.1 volume NaAc and 0.7 volume isopropanol.
Sequencing and data analysis
DNA was sequenced using Illumina GII sequencer at the Advanced Technology Center, NCI (Rockville, Md). Sequenced DNA was aligned to the mouse genome (mm9) using Eland or Bowtie . Hotspots were identified as previously described [1, 4, 28] using a Fdr of 0% and a tag density threshold at the mode. Hotspots were identified either from tag libraries generated from individual concentrations of Benzonase, Cyanase or DNase I or tag libraries pooled from different concentrations of enzymes or replicates of the same concentration. Bioinformatic analysis was performed using HOMER , Galaxy  and tools described in [4, 31]. Sequence data is accessible at GEO: GSE39982.
Previous published data used for analysis
RNA-seq ; SREBP-2 ChIP-seq peaks ; FOXA2 ChIP-seq peaks ; MNase-seq tag library (GEO: GSM717558) ; H3K4me1 and H3K4me3 ChIP-seq tag library (SRA008281) ; H3K27me2 ChIP-seq tag library (GEO: GSM751034).
ChIP combined with massive parallel sequencing
DNase I digestion of chromatin combined with massive parallel sequencing
Formaldehyde assisted identification of regulatory elements
Forkhead box A2
Histone3 lysin 4 mono-methylation
Histone3 lysine 4 tri-methylation
Histone3 lysine 27 tri-methylation
Massive parallel sequencing of RNA
Sterol Regulatory Element-Binding Protein
Tissue accessible chromatin
Transcriptional start site.
LG was supported by a research grant from the Lundbeck Foundation. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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