Analysis of Combinatorial Epigenomic States - ACS Chemical Biology

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Analysis of Combinatorial Epigenomic States Paul D Soloway ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00833 • Publication Date (Web): 11 Nov 2015 Downloaded from http://pubs.acs.org on November 13, 2015

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Analysis of Combinatorial Epigenomic States Paul D. Soloway Division of Nutritional Sciences Cornell University Ithaca, NY 14853 USA Correspondence to [email protected]

Keywords: Chromatin – DNA and DNA binding proteins found in cells. DNA modifications – covalent modifications to the DNA that do not alter the primary DNA sequence, including methyl-, hydroxymethyl-, formyl-, and carboxylcytosine. Histone modifications – covalent modifications to amino acids within histones. Chromatin code – Gene regulatory information contained within combinations of DNA and histone modifications found at a given location in the genome. Epigenome – The collective state of histone and DNA modifications in a cell. Chromatin immunoprecipitation – (ChIP) Isolation of chromatin carrying a particular histone or DNA modifications, or DNA binding proteins by immunoprecipitation. Used to map the locations of the modifications in the genome. Bisulfite sequencing – Method for assessing modification states of cytosine in DNA, variations of which enable distinguishing methyl- and hydroxymethylcytosine. Used to map the locations of the modifications in the genome. Combinatorial epigenomics – Approaches to discern the locations of combinations of DNA and/or histone modifications in the epigenome.


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Abstract: Hundreds of distinct chemical modifications to DNA and histone amino acids have been described. Regulation exerted by these so-called epigenetic marks is vital to normal development, stability of cell identity through mitosis, and non-genetic transmission of traits between generations through meiosis. Loss of this regulation contributes to many diseases. Evidence indicates epigenetic marks function in combinations, whereby a given modification has distinct effects on local genome control, depending on which additional modifications are locally present. This review summarizes emerging methods for assessing combinatorial epigenomic states, as well as challenges and opportunities for their refinement. 1. The combinatorial chromatin code The “Histone Code” hypothesis advanced by Strahl and Allis in 2000 posited that modifications to one or more N-terminal tails of histones within single or multiple nucleosomes, may act sequentially or in combination to elicit downstream genomic regulatory events. Mechanistically, it was proposed that this occurs in collaboration with factors capable of reading the code through modification-specific binding 1. At the time these ideas were advanced, the known modifications were largely restricted to acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation; and the known amino acid residues harboring the histone modifications were likewise fewer than 20. Since then, there has been much support for the histone code hypothesis, and in particular, that combinations of modifications to both DNA and histones, and not simply single modifications to histones by themselves, are vital to the mechanisms by which chromatin enables genomic regulation. Among the evidence supporting the histone code, or more broadly, a chromatin or epigenomic code, are the following: First, mass spectrometric analysis revealed that individual histones do in fact carry multiple modifications on many peptide fragments analyzed 2-6. Though unstructured histone tails are especially richly modified [reviewed in 7], globular domains, surfaces at the DNA entry and exit sites, and residues near the dyad axis also harbor modifications [reviewed in 8]. There are at least 20, distinct covalent adducts found on histones, and 128 amino acid residues in the four core histones (H2A, H2B, H3 and H3) harboring modifications [reviewed in 9]. Greater than 99.9% of the histone variant H3.2 species found in HeLa cells carry multiple modifications 4. Second, chromatin regulatory factors that include so-called writers, readers and erasers of epigenomic marks, bind histones through at least 20 characterized reader domains [reviewed in 10, 11 ]. Their binding and activity is commonly sensitive to the modification states of nearby amino acids on the bound histones 12-19. Additionally, protein reader domains are known that regulate DNA binding in a manner sensitive to DNA modification states. These are fewer in number than the histone readers, and include the MBD, SRA, BTB/BOZ and CxxC domains. Interestingly, some readers have domains recognizing both DNA and histone modification states, molecularly coupling regulation afforded by these two classes of chromatin modifications [reviewed in 20]. When multiple distinct modification-binding domains are present on reader proteins, their capacity to respond to the modification states on the bound and nearby histones is increased. Third, it is known that distinct epigenomic regulatory mechanisms exhibit cross-talk, for example between cytosine methylation and histone deacetylation 21, 22, and cytosine methylation and histone methylation 23. This invites broadening the concept of the histone code to the chromatin or epigenomic code. The potential complexity of the chromatin code increases exponentially with increases in the number of known reader proteins 10, 11, histone modifications that were already described, and DNA modifications including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and, 5-carboxylcytosine (5caC) 24, 25, and possibly N-methylated nucleotides (i.e. N3-methylcytosine, N3-methylthymine, N1-methyladenine) and 5hydroxymethyluracil – in addition to 5-methylcytosine (5mC). Figure 1 depicts a manifestation of the histone code where a nucleosome harboring modifications of its histones and DNA, recruits a reader protein whose binding requires two histone modifications; and the reader in turn recruits effectors responsible for downstream chromatin based reactions. ACS Paragon Plus Environment


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2. Widely-used analyses of single epigenomic features Most analyses of epigenomic states query single epigenomic features at a time, and accordingly, the richness of information encoded by combinations of features may easily be overlooked. To characterize genomic locations of individual histone modifications in chromatin samples, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been, and continues to be the most widely used method, subject to the availability of effective antibodies specific for histone modifications of interest 26. Antibodies have also been used to enrich for DNA harboring the modified nucleotides 5mC 27, 5hmC 28-30, 5fC 31, and 5caC 32. Affinity methods do not provide single nucleotide resolution maps of modified bases; this level of resolution is achieved by methods that use chemical, enzymatic, or both approaches to further modify bases harboring modifications of interest, and alter the primary DNA sequence in a manner that is diagnostic of the underlying base modification. Bisulfite sequencing identifies locations of methylated and hydroxymethylated bases, without distinguishing them. Variations of this technique including TAB-seq 33 and oxBS-seq 34 provide locations of 5hmC and mC respectively; and MAB-seq provides locations of 5fC and 5caC in CpG dinucleotides 35. Additional methods combine chemical modification of DNA followed by affinity capture of the modified sequences to map 5hmC 36, 37 and 5fC 38. TAB-seq, oxBS-seq and MAB-seq require splitting samples for two parallel analyses to infer locations of modified nucleotides. Many rich epigenomic datasets include ChIP-seq analyses performed with a variety of antibodies, and characterizations of different base modifications, with each assay performed in parallel. When these data are superimposed, it becomes clear where distinct epigenomic features do not reside together, though individual genomic loci are commonly seen to exhibit evidence of multiple epigenomic features. In such cases, it is tempting to infer that the features are simultaneously present at those loci in the cells and tissues analyzed, but this inference is only rarely experimentally validated 39, 40. One can imagine that even for a clonally derived cell line, subpopulations of cells exist, and that a given locus in one population has a distinct epigenomic status in another population. In such cases, when datasets are superimposed, an average state is reported that might never exist in any one population. It is not clear how big a problem this is. Statistical strategies have been combined with single ChIP experiments to identify sites where combinations of chromatin features are likely to be found (reviewed in 41). Experimental methods that directly assay combinations of epigenomic features can eliminate some of the ambiguity and minimize the need for probabilistic inferences. A few methods have been developed that facilitate more direct analyses of combinations of epigenomic marks. These, however, are not widely used for at least two reasons. First, the methods for combinatorial analyses are still under development, and undergoing improvements in efficacy and cost. Second, single mark analyses continue to provide valuable and previously unrecognized insights about genomic regulation during development and in disease states. Accordingly, many investigators do not yet feel compelled to refine their epigenomic studies to include combinatorial analyses. Nonetheless, there are many compelling questions that will be best addressed by such approaches. For example, several lines of evidence indicate that 5mC and H3K27me3 are mutually exclusive in normal primary cells, but are mutually dependent in immortalized cells 4247 . This raises the possibility that loci where they are coordinately placed in cancers might be important to disease etiology, and that identifying those loci might provide insights into disease mechanisms. Advances that refine current epigenomic analytical tools to enable true combinatorial analyses of multiple epigenomic marks will provide richer insights into the extent of epigenomic variation necessary to evaluate its biological importance. The remainder of this review will discuss existing and emerging technologies for combinatorial mark analyses, as well as persisting challenges that will be important to overcome. Table 1 summarizes the methods described, which are displayed pictorially in figure 2. 3. ChIP-BS-seq By integrating ChIP-seq with bisulfite sequencing, it is possible to map both the genomic locations of single histone modifications, and to characterize the underlying 5mC/5hmC states at sites harboring the histone modifications 40, 48 (Figure 2A). In principle, this strategy can be applied to DNA isolated from any ChIP ACS Paragon Plus Environment

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experiment, and instead of using BS-seq, rely on oxBS-seq, TAB-seq, or MAB-seq query 5mC, 5hmC or 5fC/5caC separately, provided that sufficient amounts of DNA are recovered from ChIP. The amount required for such combinatorial analyses are higher than for ChIP alone, because of additional processing of recovered DNA required to discern DNA modifications, and also because of the need for deeper sequencing to map DNAs whose complexity is reduced by the chemistries of each method 49, 50. Improvements to BS-seq methods have enabled 5mC/5hmC characterizations of single cells 51 and can be applied to DNA isolated by prior ChIP. Additionally, emerging sequencing methods that indirectly detect modification state of the DNA templates sequenced (see below 52-54) might eliminate the need for the additional processing or deeper sequencing required when implementing bisulfite based methods. 4. Alternate sequencing technologies There are two disadvantages of ChIP-BS-seq for combinatorial analysis of histone modifications and 5mC placement. First, bisulfite treatment damages DNA leading to loss of precious material recovered from ChIP. This will limit library read depth and accuracy of the resulting epigenomic maps. Second, by converting unmethylated cytosines to uracil, bisulfite treatment reduces the complexity of the DNA; cytosines may be read as C or T residues during sequencing. This makes unambiguous alignment of reads to the genome less common, and requires greater read depth when preparing maps of DNA modification states. However, the Pacific Biosciences and pending Oxford Nanopore single molecule sequencing platforms, which are distinct from the Illumina platforms, hold promise for bypassing both limitations, enabling the read out of the modification states in DNA isolated from ChIP. Two features of these platforms make this possible. First, both of the single molecule methods can be used to sequence DNA templates that are naturally existing in the cells of interest. With this workflow, the DNAs are not generated by amplification after ChIP, and the modification states of the DNAs applied to the platforms are preserved. Second, during sequencing, modified nucleotides have been shown to exhibit distinct behaviors from unmodified nucleotides, which enables their modification states to be inferred. With the Pacific Biosciences SMRT platform, sequence data are determined by detecting incorporation of fluorescent nucleotides during DNA synthesis guided by the naturally occurring template. The kinetics of incorporation of nucleotides is influenced by adjacent 5mC 52 and 5hmC 53 residues. Nanoporebased platforms have been used to distinguish C, 5mC, hmC, fC and caC 55, 56 though error rates can be high 57 . In principle, pending further advancements with nanopore sequencing, either of these single molecule sequencing approaches can report coincidence of DNA and chromatin modifications, if DNA isolated by ChIP is sequenced on these platforms. 5. Sequential or re-ChIP Sequential- or re-ChIP uses chromatin immunoprecipitated with one antibody and subjects it to reprecipitation with a second antibody before analyzing the DNA 58-60 (Figure 2B). Re-ChIP, was used to demonstrate that the so-called bivalent state composed of H3K4me3 and H3K27me3, detected by single ChIP studies on lineage specific genes in pluripotent stem cells, were actually simultaneously present on the same nucleosome 39, 61. Re-ChIP, is not commonly used, particularly for genome wide studies, in part because single ChIP remains to be informative. As a practical matter, a genome wide re-ChIP using standard protocols would require very large inputs of chromatin and antibodies because chromatin precipitation is inefficient. Furthermore, many re-ChIP protocols require detergent release of chromatin after the first ChIP, and dilution prior to the second; both steps will necessarily limit efficiency of the second antibody binding reaction. Some promising strategies, however, might overcome these barriers. Microfluidic devices using small reaction volumes have enabled use of very low inputs of cellular chromatin for genome wide analyses, while keeping chromatin concentration and capture reagents high. These have improved overall ChIP efficiency, and might facilitate re-ChIP as well 62, 63. Library preparation improvements that were developed for increasing throughput of single ChIP experiments might also enable more effective re-ChIP processing 64. Additionally, use of photocleavable linkages to liberate chromatin from the first antibody in a reChIP study would minimize ACS Paragon Plus Environment


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the inefficiencies imposed by detergents. Finally, if DNAs from re-ChIP studies were sequenced using the single molecule platforms described earlier, this could, in principle, reveal the DNA modification states of the underlying DNA. 6. Mass spectrometry (MS) Proteomic analyses of histone peptides described above have provided the most unequivocal evidence for the existence of specific histone modifications and also for the plurality of modifications on individual histones. Three main MS based strategies have been used for histone analyses (reviewed in 65, 66. In the bottom up approach, histones are fragmented into peptides, typically by trypsin digestion, limited by prior histone derivatization. After reverse phase high performance liquid chromatography purification, and often further fragmentation by inert gas collision, highly sensitive identification and quantification of histone modifications by MS is possible. Because peptides resulting from enzymatic and physical fragmentation are short, combinations of histone modifications are rarely detected. This limitation is overcome by middle down and top down approaches, which forgo digestion by trypsin that cuts histones frequently. Middle down uses proteinases cutting infrequently in histones, with peptide purification often by hydrophobic interaction chromatography; top down uses intact proteins. In both middle and top down methods, peptides/proteins are commonly fragmented by anion-carried electrons prior to collecting spectra, which can report combinations of modifications existing on individual histones. Because fragments analyzed by middle and top down approaches are larger than those analyzed by bottom up, there is a broader distribution of charge states, and a larger number of potential histone modifications on the molecules, both of which reduce signals for any given modification or combination of modifications. Application of these MS methods to individual purified histone species had led to the identification of the plurality of adducts on specific histone residues. Their application to nucleosomes previously isolated by ChIP revealed that nucleosomes carrying H3K27 methylation, or H4K20me1 are present in forms with one or both of the respective histones modified. Furthermore, bivalent nucleosomes harboring H3K4me3 or H3K36me3 in addition to H3K27me3 exhibit the two H3 modifications on separate copies of H3, rather than on a single molecule 61. and nucleosomes associated with specific loci. By applying MS methods to chromatin retrieved from specific genomic loci, it is possible to characterize transcription factors, as well as histones and their modification states at those loci (Figure 2C). Such analyses have relied on capturing chromatin from defined loci using one of three sets of approaches. Three examples of the first set of approaches include chromatin affinity purification with MS (ChAP-MS, ), targeted chromatin purification (TChP, 680, and insertional ChIP (iChIP, 69). In each of these methods, a sequence of interest is modified in the relevant cell type to contain the DNA binding motif for a protein, which, when expressed in cells of interest, or added to chromatin isolated from those cells, enables the capture of the sequence of interest using antibody or other affinity probe specific for the DNA binding protein. One disadvantage is that these methods require genetically modifying the endogenous locus, which will not be practical to implement for studies of naturally occurring cells and tissues. A second disadvantage in some earlier applications is that the target cells must also be engineered to express the locus specific DNA binding protein.

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Three examples of the second set of approaches that bypass the requirement to modify the endogenous locus of interest include TAL-ChAP-MS 70, engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP, 71), and CRISPR -ChAP-MS 72. These use either TALENs 70 or the CRISPR/Cas9 system to capture sequences of interest. As with the first set of approaches, current versions of these methods require that cells are modified to express either a designed TALEN, or a catalytically inactive form of Cas9 (dCas9) along with a guide RNA(gRNA) specific for the locus of interest, followed by antibody capture. It is possible that the proteins and gRNAs required for sequence capture could be added to chromatin-containing lysates, bypassing the requirement for any genetic manipulations of cells and tissues prior to sequence capture. ACS Paragon Plus Environment

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In the third set of approaches, no genetic manipulations are required, instead, chromatin of interest is captured by locked nucleic acid (LNA) probes or other oligonucleotides specific for sequences of interest 73, 74. Nucleic acid hybridization capture requires denaturing sites of homology of the target chromatin. and accordingly, causes some loss of associated proteins. It is possible that polyamide nucleic acids (PNA) probes, which, because they lack negatively charged phosphates, may enable PNA invasion into negatively charged DNA duplexes without requiring prior denaturation of the duplex. Successful application of these methods have required very abundant amounts of chromatin from organisms with low complexity genomes, or highly repetitive sequences of interest from more complex genomes. This compensates for the inefficiency of the capture, although recent applications of one of these methods provided efficiency improvements 75. Until MS throughput increases enormously, successful application of these approaches will be limited to the analyses of few loci. 7. Single molecule methods The previously discussed Pacific Biosciences and nanopore-based sequencing platforms represent single molecule methods that can report the modification states of DNAs isolated from ChIP studies. Separate from these are several methods that have been developed for characterizing chromatin features on individual molecules. Because of the exquisite sensitivity inherent to single molecule methods, they provide opportunities to analyze combinatorial epigenomic states where high sensitivity is essential, especially when methods like re-ChIP provide only low recoveries of input chromatin. Most of these single molecule methods are either in the development stage, or represent opportunities for development efforts that could enable analyses of combinatorial states. There are critical limitations that must be addressed by each single molecule approach. The first relates to throughput. The time to collect epigenomic information from each single molecule assayed will influence the depth of genome coverage possible. Second, when performing analyses of multiple epigenomic marks on single chromatin molecules using fluorescent antibody binding, it is ideal to saturate available epitopes with the antibodies, ensuring all marks of potential interest are detected. This will be influenced by the dissociation constants (Kd) of the binding reactions, and off rates (Koff) for procedures that require removal of unbound antibodies. It will also be influenced by epitope masking, whereby binding of one antibody to a chromatin modification sterically hinders the binding of a second antibody to a distinct modification. Third, optimal epigenomic analyses will reveal the DNA sequences containing modifications of interest, however, many current single molecule methods either do not report the underlying sequences, or report a limited number of predetermined sequences. Three methods described below (DNA curtains, ordered chromatin arrays, and nanochannel squeezing) involve diffraction limited optical observations of immobilized chromatin fragments. Accordingly, the spatial resolution of observed features on large chromatin fragments, whether they are epigenomic marks or probes detecting specific sequences, will be on the order of 300 nm 76) or 8 kbp 77, 78), which is approximately 50 nucleosomes, if chromatin was decompacted from the 30 nm fiber. Use of shorter chromatin fragments will improve this genomic resolution, but at the cost of reduced throughput for these three methods. Several distinct single molecule methods have been developed (reviewed in 79-82). Many of these methods are currently at the proof-of-principle stage, nonetheless, further developments that will enhance their utility can be anticipated. 7.1.1. ChIP-string The NanoString nCounter platform was originally used for quantifying abundance of a limited number of RNAs without enzymatic processing of the input RNAs. The method relies on fluorescent bar-coded probes capable of uniquely-identifying transcripts of interest. When adapted to interrogate DNAs isolated after ChIP experiments (ChIP-string 83 Figure 2D), the platform reported the presence of chromatin features at 487 loci. In that study, chromatin features were assayed individually. Because of the high single molecule sensitivity afforded by this method, it may be appropriate for re-ChIP experiments and dual mark assessment, where chromatin yields are very low. An important limitation of the method is that it is not compatible with genome ACS Paragon Plus Environment


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wide analyses; only a limited number of loci can be queried, depending on the number of fluorescent bar codes available. 7.1.2.

DNA curtains

DNA curtains are prepared by anchoring the biotinylated ends of DNA to streptavidin in lipids tethered to a solid substrate in a flow cell (reviewed in 84, Figure 2E). When aqueous solutions containing fluorescent intercalators are flowed through the cell, DNA fibers elongate and can be visualized by fluorescent microscopy. These methods have been used to study factors affecting nucleosome deposition 85 and the impact of nucleosomes on DNA interactions with repair proteins 86. It might be possible to characterize where histone or DNA modifications exist on naturally occurring chromatin by biotinylating the ends of chromatin before anchoring and elongating fibers under flow conditions, and by including fluorescent probes recognizing modifications of interest. Further enhancements would be required to reveal the underlying DNA sequences, and these could include use of fluorescent probes to detect those sequences. As with sequence capture methods followed by MS, these will be limited, initially, to highly repetitive sequences in complex genomes, or to analyses of low complexity genomes. 7.1.3.

Ordered chromatin arrays

Chromatin fragments can be stretched, and held in an elongated state by devices fabricated from polydimethylsiloxane (PDMS, Figure 2F). Devices have included micron scale PDMS pillars protruding from the device surface, and that are spaced at micron scale intervals. When aqueous solutions of chromatin are placed between the device and a glass coverslip, chromatin fibers can be elongated and localized between pillars by drawing a coverslip across the device. In one study, histone H3 was detected on the elongated chromatin by binding fluorescent antibodies and imaging the devices. It might be possible to extend this method to report patterns of coincidence and mutual exclusion of chromatin features using antibodies recognizing distinct chromatin modifications, each labeled with a spectrally distinct fluorophore. The resolution will be fundamentally limited by light diffraction, and methods have not been implemented to identify the underlying DNA sequences on ordered arrays 76. 7.1.4.

Nanochannel squeezing

Chip-string, DNA curtains and ordered arrays each require immobilizing and elongating molecules prior to detecting fluorescent properties (Figure 2F). In nanochannel squeezing, chromatin is flowed into an elastic channel fabricated with PDMS, which is then narrowed to a cross section of approximately 200nm x 200nm, confining linear fragments. This can also be done using narrow rigid channels 77, 78. In one study using elastic channels to confine HeLa chromatin, fluorescent antibodies recognizing H4ac and H3K9me3 were included and domains of binding were reported to be mutually exclusive 87, consistent with expectations that these activating and silencing marks would not be coincident. In such studies, possible binding sites might not be saturated with the antibodies, and accordingly, caution is required when interpreting the data. Elastic channel confinement and squeezing was also used to characterize two histone modifications on chromatin reconstituted on T7 phage DNA 88; and, DNA methylation in experimentally methylated lambda DNA was detected using a fluorescently tagged MBD portion of MeCP2 78. 7.1.5. SCAN Single chromatin molecule analysis at the nanoscale (SCAN), is analogous in principle to flow cytometry and fluorescent activated cell sorting (FACS, Figure 2G). In the cell based assays, fluorescent antibodies are bound to a population of cells, the fluorescent properties of single cells are measured as they flow through an inspection or focal volume; and in the case of FACS, cells are isolated based on user defined properties. In SCAN, chromatin fragments are bound to fluorescent antibodies or other probes recognizing chromatin modifications; molecules are then driven by a voltage potential through a fluidic channel; and finally, fluorescent properties of individual molecules are detected. By using multiple antibodies, each recognizing distinct epigenomic marks and carrying distinct flurophores, combinations of chromatin modifications can be ACS Paragon Plus Environment

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detected and their abundances measured 47, 89. Similar to FACS, single molecules can be sorted based on their modification state 90 and, with improvements in throughput, isolated molecules can be used for downstream applications such as sequencing. 7.1.6. Droplet approaches Approaches using single molecules enclosed in droplets hold promise for epigenomic analyses (reviewed in 91, Figure 2H). Droplets with a 10µ diameter (0.5nL volume) are readily formed in microfluidic channels; they can be formed from dilute chromatin solutions to generate droplets with single molecules according to the Poisson distribution; chromatin containing droplets can merged with other droplets carrying reagents; and the fluorescent properties of droplets can measured under flow at high throughput. One can envision confining single chromatin molecules along with quenched fluorescent affinity probes to report the epigenomic states of the confined chromatin. Furthermore, droplets carrying fluorescent properties of interest can be sorted using acoustic waves 92, and DNA from collected droplets isolated for sequencing. 8. Reagent limitations, an ongoing challenge ChIP, and many of the above referenced methods for combinatorial analysis of epigenomic states rely on the availability of reliable affinity reagents that are specific and selective for chromatin features of interest. For combinatorial analyses, it is also important that the affinity reagents have binding kinetics that favor highly efficient binding, with low Kd values, high on-rates, and low off-rates. Whereas inefficient binding can still enable high-coverage, genome-wide mapping of single marks using abundant chromatin sources, for combinatorial mark analyses, the drop in coverage is proportional to the products of the binding efficiencies. Reliability of antibodies from animal sources, the most widely used affinity reagents, is an ongoing challenge 93. Even when reliable antibodies with high binding efficiencies are available for a given histone modification, their typical sizes (150kDa, and 10-15nm width) can sterically hinder binding of a second antibody to a distinct nearby modification. This can fundamentally limit combinatorial analyses requiring simultaneous binding of affinity reagents. Several approaches have been reported that hold promise for circumventing these challenges. Recombinant antibodies with minimal amino acid content have been reported with Kd values for histone modifications that are in the low double digit nM range; these are close to but still less favorable than natural antibodies. They have the added advantage of exhibiting reduced sensitivity to amino acid modifications on amino acids near to the target epitope 94. Aptamers specific for histone modifications have also been reported with KD values also in the low double digit nM range 95-97. Chemical modifications to aptamers, could provide reagents with KD values in the pM range, and slow off rates 98. An additional strategy to develop antibody alternatives relied upon use of peptides derived from naturally occurring histone modification interaction domains (HMID) from ATRX, and MPHOSPH8 99. Using these in ChIP-seq like assays to detect H3K9me3 in HepG2 cells revealed good correlation between results using a 129 amino acid ATRX HMID and commercial antibodies, though there was also evidence of off target binding by the HMID. Additionally, affinities of the HMIDs were in the high nm to low µm range, less favorable than bona fide antibodies. Nonetheless, HMIDs can be expressed as a single polypeptide in bacteria, are onetenth the size of antibodies, and provide an opportunity to express HMIDs as fusion proteins to capture combinations of marks. Reader protein modules have been assembled as fusion proteins to capture chromatin harboring combinations of histone modifications 100. Specificities that reader modules have for specific histone modifications require careful determination, and affinities tend to be only moderate, with KD values for in the low micromolar range, 1,000 fold higher than antibodies. For both HMIDs and reader protein modules, it might be possible to implement mutagenesis, selection and amplification strategies to identify modules with improved specificity, selectivity and affinity for defined combinations of histone modifications. This would be an important advance to enable both single and combinatorial epigenomic analyses. ACS Paragon Plus Environment


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9. Closing remarks It is clear that information carried within chromatin, in addition to the underlying genomic sequences, control phenotypes. Epigenomic profiling by ChIP-seq and bisulfite based methods have revealed chromatin features important for that control, but it is almost certain that current understanding is still quite rudimentary. The plurality of known chromatin modifications, many of which have only been recently discovered, and the strong evidence that these can work in combination with each other to effect genome regulation, provide motivation for characterizing where combinations of features truly reside in the genome, and the regulation those combinations provide. Though current technologies remain very informative, methods for combinatorial analyses, perhaps including next generation embodiments of those described, here hold promise for providing a more nuanced and refined view of the epigenome and the regulation it affords.

Acknowledgements: Funding support from NIH HG006850.


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Figure Legends. Figure 1. Combinatorial epigenomic states and genomic regulation. Depiction of a nucleosome with two copies of each of the four core histones (colored balloons) that carry modified amino acids (diamonds), and DNA harboring cytosine modifications (mC, hmC, fC and caC). Hypothetical reader protein (rectangle) with a plant homeodomain (PHD), and a bromodomain (BRD) capable of binding methylated and acetylated amino acids respectively. Additional interacting effector proteins (oval) may be recruited and participate chromatin based reactions including RNA transcription, DNA replication and repair, and others. Readers may bridge distinct histones, and interact with the DNA in a manner sensitive to DNA modification states. Figure 2. Strategies and workflows for combinatorial epigenomic analyses. Many of the approaches depicted are in the development phase, or are logical extensions of developed technologies. See text for further descriptions and citations. A. ChIP-mC-seq identifies regions of the genome harboring specific histone modifications, and the underlying DNA methylation state, which may be determined by traditional bisulfite sequencing, SMRT-sequencing using the Pacific Biosciences platform, or emerging nanopore based methods. B. Re-ChIP identifies regions of the genome harboring combinations of histone modifications. Chromatin isolated by a first immunoprecipitation may be freed from the first antibody by detergents and then diluted, or freed by light if using antibodies with photocleavable linkages. Recovered chromatin is the input for a second immunoprecipitation. C. Sequence capture-MS applies mass spectrometric analysis to chromatin captured using sequence-specific DNA binding proteins or oligonucleotides (double-headed arrow). MS can also be applied to chromatin recovered by ChIP, revealing modifications found along with that detected by the antibody used. However, the underlying DNA sequences are not revealed. D. ChIP-string uses the Nanostring platform, and barcoded fluorescent probes (short colored segments) to identify sequences recovered by ChIP. Barcode limitations do not enable genome wide coverage, but the sensitivity afforded by this method may be amenable to Re-ChIP studies. E. DNA curtains prepared by biotinylating DNA ends (B), binding material to streptavidin (SA) localized in lipid bilayers (wavy lines) can be elongated and fluorescently imaged under flow conditions that elongate the tethered DNA. Application of this method to chromatin, and use of fluorescent antibodies recognizing chromatin modifications may reveal combinatorial chromatin states. F. Ordered arrays and nanochannel squeezing respectively tether or confine chromatin to nanoscale structures fabricated from polydimethylsiloxane (blue structure at bottom), where fluorescent affinity reagents detecting chromatin modifications can be bound, revealing combinatorial chromatin states. G. In SCAN chromatin molecules are driven through nanoscale fluidic channels along a voltage gradient. Fluorescent affinity reagents reveal molecules harboring combinations of chromatin modifications as they flow through an inspection volume illuminated by excitation lasers (parabolic shape). The method requires detectors for emitted fluorescence, dilute solutions to ensure single molecule detection, and DNA intercalators to identify chromatin fragments, which may be bound to fluorescent affinity reagents. H. Chromatin confined to aqueous droplets, 1-10µm in diameter, flowing in an oil phase in micron scale channels can be fused with other droplets carrying reagents. In the example shown, a fluorescent affinity reagent (1 and 2) with quenching moieties attached (Q) emit fluorescence when bound to chromatin modifications. Histones, colored balloons; diamonds, histone modifications; mC, methylated cytosines and its oxidation products; numbered stick figures, antibodies and other affinity probes recognizing histone and DNA modifications, where blue and red figures depict fluorescently tagged reagents and wavy arrows depict their emissions.



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Table 1. Methods for single and combinatorial epigenomic analyses

Single Mark Analyses

Feature measured

Information reported

Limitations

Histone modifications/ chromatin binding proteins

Genomic locations of individual chromatin features

Coincidence of multiple marks inferred; statistical methods to improve inferences exist

BS-seq

5mC/5hmC

Genomic locations of 5mC/5hmC

Does not distinguish 5mC from 5hmC, or among C, 5fC and 5caC on same template

oxBS-seq

5mC

Genomic locations of 5mC

Does not distinguish among C, 5hmC, 5fC or 5caC on same template

TAB-seq

5hmC

Genomic locations of 5hmC

Does not distinguish among C, 5mC, 5fC or 5caC on same template

MAB-seq

5fC/5caC

Genomic locations of 5fC/5caC

Does not distinguish 5fC from 5caC, among C, 5mC or 5hmC on same template

ChIP-string

Chromatin binding factors

Which of up to 800 genomic locations bind feature of interest

Does not provide full genome coverage

Features measured


Limitations

Histone modifications/ chromatin binding proteins

Genomic locations of combinations of chromatin features

Inefficiencies of both ChIP steps limits genomic coverage.

Histone modifications or Chromatin binding proteins


ChIP-seq

Combinatorial Mark Analyses – Established methods re-ChIP or sequential ChIP

ChIP-BS-seq

Requires high quality affinity reagents



Requires implementing BS-seq, oxBS-seq, TAB-seq, or MAB-seq to DNA isolated from ChIP Requires deeper sequencing than single ChIP for effective mapping due to reduced DNA complexity resulting from bisulfite and other chemistries

C and or modified forms of C

ChIP-SMRT-seq



Requires sequencing ChIP DNA on Pacific Biosciences SMRT sequencing, with kinetic measures of nucleotide incorporation

Abundance, identity and coincidence of multiple histone modifications

Requires modifications reside on nearby amino acids on the same histone, unless analyte is from a prior ChIP

C, 5mC, 5hmC Mass spectrometry

Histone modifications

Does not report genomic locations of histone modifications Combinatorial Mark Analyses – Emerging methods ChIP-nanopore-seq

Features measured


Further advances needed



Requires sequencing ChIP DNA on nanopore sequencing platforms with current measurements that distinguish modified from unmodified C

C and or modified forms of C


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ChIP-string

ACS Chemical Biology Chromatin binding factors

Which of up to 800 genomic locations bind feature of interest

Requires input from re-ChIP and combinations of high quality affinity reagents Does not provide full genome coverage

DNA curtains

Ordered chromatin arrays

DNA binding proteins

Binding properties and dynamics of interactions between DNA binding proteins defined DNA substrates

Not yet adapted for naturally occurring chromatin

Histones

Presence of histones on chromatin; readily adaptable to detect histone modifications

Does not report sequence of underlying DNA

Requires binding chromatin with combinations of high quality affinity reagents

Requires binding chromatin with combinations of high quality affinity reagents Low resolution between detected features

Chromatin elongated by confinement

Histone modifications and 5mC

Presence of histone modifications and 5mC

Does not report sequence of underlying DNA Requires binding chromatin with combinations of high quality affinity reagents Low resolution between detected features

SCAN

Histone modifications and 5mC, individually or in combination

Abundance of chromatin features Can be run in analytical or preparative modes


Requires binding chromatin with combinations of high quality affinity reagents Limited molecule per minute throughput


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Figure 1. Combinatorial epigenomic states and genomic regulation. Depiction of a nucleosome with two copies of each of the four core histones (colored balloons) that carry modified amino acids (diamonds), and DNA harboring cytosine modifications (mC, hmC, fC and caC). Hypothetical reader protein (rectangle) with a plant homeodomain (PHD), and a bromodomain (BRD) capable of binding methylated and acetylated amino acids respectively. Additional interacting effector proteins (oval) may be recruited and participate chromatin based reactions including RNA transcription, DNA replication and repair, and others. Readers may bridge distinct histones, and interact with the DNA in a manner sensitive to DNA modification states. 254x338mm (72 x 72 DPI)



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Figure 2. Strategies and workflows for combinatorial epigenomic analyses. Many of the approaches depicted are in the development phase, or are logical extensions of developed technologies. See text for further descriptions and citations. A. ChIP-mC-seq identifies regions of the genome harboring specific histone modifications, and the underlying DNA methylation state, which may be determined by traditional bisulfite sequencing, SMRT-sequencing using the Pacific Biosciences platform, or emerging nanopore based methods. B. Re-ChIP identifies regions of the genome harboring combinations of histone modifications. Chromatin isolated by a first immunoprecipitation may be freed from the first antibody by detergents and then diluted, or freed by light if using antibodies with photocleavable linkages. Recovered chromatin is the input for a second immunoprecipitation. C. Sequence capture-MS applies mass spectrometric analysis to chromatin captured using sequence-specific DNA binding proteins or oligonucleotides (double-headed arrow). MS can also be applied to chromatin recovered by ChIP, revealing modifications found along with that detected by the antibody used. However, the underlying DNA sequences are not revealed. D. ChIP-string uses the Nanostring platform, and barcoded fluorescent probes (short


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colored segments) to identify sequences recovered by ChIP. Barcode limitations do not enable genome wide coverage, but the sensitivity afforded by this method may be amenable to Re-ChIP studies. E. DNA curtains prepared by biotinylating DNA ends (B), binding material to streptavidin (SA) localized in lipid bilayers (wavy lines) can be elongated and fluorescently imaged under flow conditions that elongate the tethered DNA. Application of this method to chromatin, and use of fluorescent antibodies recognizing chromatin modifications may reveal combinatorial chromatin states. F. Ordered arrays and nanochannel squeezing respectively tether or confine chromatin to nanoscale structures fabricated from polydimethylsiloxane (blue structure at bottom), where fluorescent affinity reagents detecting chromatin modifications can be bound, revealing combinatorial chromatin states. G. In SCAN chromatin molecules are driven through nanoscale fluidic channels along a voltage gradient. Fluorescent affinity reagents reveal molecules harboring combinations of chromatin modifications as they flow through an inspection volume illuminated by excitation lasers (parabolic shape). The method requires detectors for emitted fluorescence, dilute solutions to ensure single molecule detection, and DNA intercalators to identify chromatin fragments, which may be bound to fluorescent affinity reagents. H. Chromatin confined to aqueous droplets, 1-10µm in diameter, flowing in an oil phase in micron scale channels can be fused with other droplets carrying reagents. In the example shown, a fluorescent affinity reagent (1 and 2) with quenching moieties attached (Q) emit fluorescence when bound to chromatin modifications. Histones, colored 254x338mm (72 x 72 DPI)


Analysis of Combinatorial Epigenomic States - ACS Chemical Biology

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