Dynamic Chromatin Regulation from a Single Molecule Perspective

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Dynamic Chromatin Regulation from a Single Molecule Perspective Beat Fierz* Laboratory of Biophysical Chemistry of Macromolecules, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland ABSTRACT: Chromatin regulatory processes, like all biological reactions, are dynamic and stochastic in nature but can give rise to stable and inheritable changes in gene expression patterns. A molecular understanding of those processes is key for fundamental biological insight into gene regulation, epigenetic inheritance, lineage determination, and therapeutic intervention in the case of disease. In recent years, great progress has been made in identifying important molecular players involved in key chromatin regulatory pathways. Conversely, we are only beginning to understand the dynamic interplay between protein effectors, transcription factors, and the chromatin substrate itself. Single-molecule approaches employing both highly defined chromatin substrates in vitro, as well as direct observation of complex regulatory processes in vivo, open new avenues for a molecular view of chromatin regulation. This review highlights recent applications of single-molecule methods and related techniques to investigate fundamental chromatin regulatory processes.

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expression, cell differentiation, and development. The whole of chromatin-associated proteins, or effectors, such as transcription factors (TF), remodelers, histone modifying enzymes, transcription activators, or repressors, as well as the location specific histone and DNA modification patterns define a chromatin state.8−10 Chromatin states are associated with a distinct chromatin structure (i.e., open and accessible or densely packed), subnuclear localization, as well as with specific transcriptional outcomes. Furthermore, they are actively maintained and can be inherited to daughter cells.5 The available dissociation constants for reader proteins with histone PTM are relatively weak, in the mid to high micromolar range.11 In agreement, direct observations by fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) demonstrate transient and highly dynamic interactions.12−14 For a molecular description and, in extension, for quantitative modeling of chromatin state organization, maintenance, and modulation, the dynamic properties of chromatin itself and its interactors have to be accurately determined. Recent progress in the reconstitution of synthetically modified chromatin,15 single-molecule biophysics, and single-cell measurements allow the investigation of fundamental processes on the molecular scale in real time. Single-molecule studies, in particular, have the advantage of eliminating ensemble averaging, thereby exposing rare events, enabling the study of heterogeneous populations and allowing direct observations of complex biological processes. In the following, I will review notable studies on fundamental chromatin regulatory mechanisms, with a focus on single-

n all eukaryotes, a large number of dynamic signaling processes feed into the regulation of genome organization and function. While the molecular processes are stochastic on the single-cell level, the establishment of stable gene expression patterns is of key importance, as this lies at the heart of cell lineage determination. These patterns have to persist in the face of large scale variations in cellular conditions, as when the cell progresses through the cell cycle or recovers from DNA damage. A far cry from inert packing material, chromatin, the nucleoprotein complex which organizes the eukaryotic DNA, plays a key regulatory role in all processes requiring access to the DNA.1 At its most basic, chromatin is composed of strings of nucleosomes (each containing two copies of the histones H2A, H2B, H3, and H4) that organize around 147 base pairs (bp) of DNA and are connected by 10−50 bp of linker DNA.2 The presence of chromatin severely restricts the biochemical accessibility of DNA. Therefore, the positioning of nucleosomes in the cell is dynamic and subject to perpetual remodeling by ATP dependent enzyme complexes.3 Indeed, genome wide mapping of DNA accessibility reveals large local variations, with highly accessible “hotspots” at active promoters and enhancers, which are often characterized by lowered nucleosome occupancy.4 Chromatin functions as a signaling hub to regulate gene expression and as a carrier of epigenetic memory,5 i.e. the establishment of stable and inheritable expression profiles that are not linked to alteration in DNA sequence. A large number of chromatin associated proteins and protein complexes interact with or catalyze the deposition as well as the removal of multiple chemical modifications of chromatin. Such modifications include methylation and hydroxymethylation of CpG dinucleotides in DNA6 as well as combinatorial patterns of post-translational modifications (PTMs) on histone proteins,7 both of which are involved in modulating gene © XXXX American Chemical Society

Special Issue: Epigenetics Received: October 12, 2015 Accepted: November 13, 2015

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DOI: 10.1021/acschembio.5b00832 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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NUCLEOSOME DYNAMICS GOVERN DNA ACCESSIBILITY The fundamental unit of chromatin, the nucleosome, forms the main barrier for DNA transactions, as tight interactions between the histone proteins and the nucleosomal DNA preclude access for many chromatin factors.16 In recent years, however, an increasing amount of data demonstrated that nucleosomes themselves are dynamic complexes.17,18 Kinetic rate constants of DNA breathing, i.e. the spontaneous unwrapping of nucleosomal DNA, were initially measured by Li and co-workers in 2005 in a fluorescence resonance energy transfer (FRET) assay19 (Figure 1a), using kinetic capture with a TF (LexA) and a combination of stopped-flow and FCS methods. They determined that DNA is partially exposed from the nucleosome core with a rate constant of about 4 s−1 and rapidly rewraps within 10−50 ms. These transient unwrapping events proved sufficient for TFs to bind to their target sequence within the nucleosomal DNA, resulting in a stabilization of the partially unwrapped state.17,19 In the following, single-molecule FRET (smFRET) experiments by van Noort and colleagues confirmed the time scale of spontaneous unwrapping20 and explored the influence of solvent conditionsin particular the ionic strengthon dynamic site exposure.21 Moreover, Wei et al. detected even faster nucleosomal DNA fluctuations on the 0.1−1 ms time scale.22 Together with the characterization of internal conformational dynamics, including H2A−H2B dimer splitting23 and slow nucleosome DNA rearrangements called “gaping,”24 smFRET studies contributed to a picture of the nucleosome as a complex particle undergoing multiscale dynamics and thus providing multiple windows of opportunity for interactions with effector proteins. Force spectroscopy provides an alternative method to investigate chromatin structure and dynamics,25−30 and as nucleosomes are obstacles to be overcome by motor proteins such as DNA polymerase, the encountered forces are highly biologically relevant. Experiments focused on the determination of the force required to disrupt nucleosomes allowed the calculation of the strength of histone-DNA contacts and revealed that partial DNA unwrapping was reversible.27,29,30 Employing optical tweezers to unzip DNA strands, and thereby displacing them from a nucleosome, Wang and co-workers could detect defined energy barriers resisting nucleosome unwrapping.30 The strongest interactions occur at the nucleosome dyad, and two smaller barriers exist at positions +40 and −40 bp from the dyad. Partial unwrapping of nucleosomal DNA thus is most likely is most frequent for DNA segments preceding those barriers, whereas nucleosomes disassemble when DNA contacts at the dyad are broken.30 Variants of the canonical histones modulate the stability of nucleosomes. Using ensemble FRET methods, the H2A variant H2A.Z has been determined to stabilize nucleosome structure,31 and force spectroscopy approaches confirmed a higher probability for H2A.Z containing nucleosomes to remain on chromatin arrays under tension.32 In contrast, the centromeric variant of H3 (Cse4 in budding yeast) exhibits weakened DNA interactions, as observed in DNA unzipping experiments.33

Figure 1. Dynamics in nucleosomes. (a) FRET based assay to study DNA breathing motions:17 Tightly DNA-wrapped nucleosomes exhibit high FRET. A TF (LexA) stabilizes the spontaneously unwrapped DNA, resulting in low FRET. Data analysis by kinetic modeling allows the extraction of rate constants for DNA unwrapping and wrapping (kopen, kclose) and TF binding and release (kon, koff). (b) A combined FRET and force spectroscopy experiment enables determination of sequence-dependent differences in DNA wrapping.35 When ramping up the pulling force, nucleosome opening is indicated by a drop in the FRET efficiency (EFRET). Lowering the force results in rewrapping and high FRET. (c) DNA based allostery:45 The glucocorticoid receptor DNA binding domain (GRDBD) binds to its consensus site that is placed at a distance L from either one or two nucleosomes. Its off-rate constant strongly depends on the distance to the nucleosomes with a maximum at 20 bp. (d) A FRET-based assay to monitor nucleosome remodeling:49,50 A FRET donor dye is attached to H2A, whereas the acceptor is positioned on the DNA. Nucleosome remodeling by ISWI remodelers results in a change in FRET that reports on the nucleosome position with bp resolution and allows the direct monitoring of remodeling dynamics.

Nucleosomal contacts are also strongly dependent on DNA sequence.34 Natural DNA sequences, which carry genetic information, generate nonsymmetric complexes when wrapped around nucleosomes. Ha and co-workers developed an elegant assay to determine conformational transitions in nucleosomes by smFRET while keeping the nucleosomes under tension using optical tweezers35 (Figure 1b). These experiments demonstrated that nucleosomes unwrap in an asymmetric fashion dependent on the DNA sequence. Within the nucleosome structure, DNA is highly bent,36 exhibiting very strong curvature compared to its overall persistence length.37 Stiffer DNA sequences require more energy to bend, thus they are bound less tightly to the nucleosome.38 Ngo et al. indeed observed that unwrapping occurs with greater probability from the side containing the stiffer DNA segments. Intriguingly, upon detachment of the DNA at one side, histone−DNA B


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methods provide an alternative approach to directly observe remodeler action. Zhuang and co-workers developed an elegant FRET-based assay that allows following nucleosome remodeling with single bp resolution49,50 (Figure 1d). They used this assay to study remodelers of the initiation switch (ISWI) family and found that DNA is translocated in steps of around 3 bp (composed of single-bp elementary steps) around the nucleosome.50 Importantly, these remodelers are able to move the nucleosome bidirectionally, resulting in ATPdependent oscillatory movements that equalize positioning in a nucleosome array.49 Depending on the remodeler type, DNA bound protein factors can influence nucleosome positioning. Mapping nucleosome positions by DNA unzipping using optical tweezers revealed that ISWI remodelers stopped at a bound TF, whereas SWI/SNF translocated nucleosomes through the factor, resulting in TF dissociation.51 As a consequence, nucleosome positions and remodeling are of critical importance for TF dynamics in a chromatin environment.

interactions at the other side are stabilized, restricting access to those sequences and stabilizing the overall nucleosome integrity. Thus, differences in local DNA flexibility, which occur in most nucleosomes in the genome, are amplified within the core particle, resulting in dynamic asymmetry. As potential downstream biological effects, RNA polymerase passage may be critically influenced by positioned asymmetric nucleosomes, and partially unwrapped nucleosomes may be stabilized throughout RNA polymerase passage. Based on these findings, it is evident that the thermodynamics and kinetics of effector−DNA interaction are altered by the presence of chromatin and are coupled to intrinsic motions within the nucleosomes themselves. TF dynamics lie at the core of transcriptional regulation, thus the on and off rates of these proteins within a chromatin context are of particular interest. In vitro, TFs can bind to naked consensus DNA with residence times in the minutes to hours,39 quite in contrast to observed in vivo dissociation kinetics with residence times in the seconds time regime.40 To shed light into TF dynamics within chromatin, Luo et al. immobilized mono- or dinucleosomes containing a TF consensus sequence near the DNA entry site and monitored transient binding events by single-molecule total internal reflection fluorescence (smTIRF) microscopy.41 Interestingly, they detected that the presence of a nucleosome not only reduced on rates but also critically lowered TF residence times, by about 2 orders of magnitude. This effect may be explained by two possible models: First, distortions in the nucleosomal DNA might reduce the binding energy of TFs, in a mechanism similar to allosteric effects in proteins. Second, dynamic competition of histones and TFs for the nucleosomes may increase the probability of TF eviction, as soon as initial DNA contacts are broken. The latter mechanism, a process termed “facilitated dissociation,” is often observed for competing DNA binding proteins.42−44 Concerning DNA mediated allosteric effects, recent results demonstrated that the effect of nucleosomes on TF dynamics does reach far into the linker DNA. Kim et al. used smTIRF microscopy to measure the residence time of a TF as a function of a nearby bound second protein factor, or indeed a nucleosome45 (Figure 1c). Remarkably, they found an oscillatory pattern in the binding free energy of the TF, as well as on its residence time, dependent on the distance of the binding site from the secondary DNA bound complex, due to distortions in the DNA. For a TF binding in between two nucleosomes, such “allostery through DNA” varied the dissociation time by up to 7 fold. TF interaction kinetics thus depend not only on the binding site sequence but also on its position relative to nucleosomes. At promoters and enhancers, nucleosomes often occupy defined positions, due to the underlying DNA sequence46 and the activity of diverse remodeling complexes. Chromatin remodelers are able to shift nucleosomes, to exchange histone variants or to evict histones or entire nucleosomes, while consuming ATP.3 Single-molecule methods, in particular force spectroscopy, have played a key role in investigating the mode of action of remodeling complexes.47 Focusing on studies in a chromatin context, notably Zhang et al. employed optical tweezers to measure the remodeling activity of Switch/Sucrose Non-Fermentable (SWI/SNF) in real time on a single nucleosome substrate.48 They determined that remodelers are actual molecular machines that produce a force of around 12 pN to translocate DNA around the histone core, thereby shifting nucleosome positioning on the genome. FRET-based

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CHROMATIN DYNAMICS ARE MODULATED BY HISTONE PTMS DNA methylation and histone PTMs provide the cell with a means of regulating chromatin dynamics. Methylation of CpG dinucleotides is mostly associated with repression; thus it is conceivable that the modification itself might alter the nucleosome structure. Indeed, smFRET and anisotropy measurements revealed that DNA methylation resulted in nucleosomes that stochastially populated a more compact and rigid state at a higher probability than nonmethylated nucleosomes.52 It remains to be seen if such effects, intrinsically affecting nucleosome dynamics and positioning, are of prime importance for genomic repression, as compared to recruitment of methyl-binding proteins and modulation of the binding affinities of other DNA binding proteins.6 Conversely, histone PTMs can affect protein−DNA interactions and therefore may have direct energetic consequences. With regards to gene regulation, lysine acetylation (Kac) marks, some of the most widespread PTM found in histones, are generally associated with euchromatin and active genes. All histones contain multiple lysine residues that can be acetylated, which result in site-specific and additive effects, either by modulating histone DNA interactions or by providing binding sites for reader proteins, i.e., bromodomains.53 Finally, lysine acetylation marks generally exhibit a high turnover.54 Lysine mono-, di-, or trimethylation (Kme1/2/3) represent a second important class of PTMs.55 Lysine methyl marks exhibit, through interactions with methylation state-specific reader proteins, a large variety of functions including distinct regulatory roles both in gene repression or activation. Do histone PTMs directly modulate nucleosome dynamics and, if so, how? A number of studies addressed this question, employing both chemical and biochemical means to generate site-specifically modified chromatin. Langowski and colleagues nonspecifically acetylated histones by treatment with acetyl phosphate and reconstituted nucleosomes. Subsequent ensemble and smFRET studies revealed modification specific conformational subpopulations, in particular an increase in open conformations within acetylated nucleosomes.56,57 In a related study, Lee et al.58 used smFRET to study the modulation of nucleosome structure through histone acetylation by the lysine acetyltransferase (KAT) Piccolo NuA4, which is specific for lysines in the H4 tail. Acetylation of the histones C


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Figure 2. Histone PTMs modulate nucleosome dynamics. (a) Introduction of acetyllysine into histones by genomic code expansion for the production of H3K56ac.62 H3K56 induces increased DNA breathing as observed by smFRET. (b) Synthetic pathway to produce modified H3, e.g., H3K56ac.65 An initial NCL reaction is followed by opening of the N-terminal thiazolidine (1), followed by a second NCL reaction (2). A final desulfurization step (3) yields native H3K56ac. (c) Magnetic tweezers are employed to stretch modified oligo-nucleosomes to determine PTM effects on dynamics and stability.66 (d) Together with H3K56ac, also H4(K77ac/K79ac) increases DNA breathing and Y41ph strengthens this effect, whereas H3(K115ac/K122ac) decreases DNA binding at the nucleosome dyad.66,68

peripheral DNA sites.66,67 Interestingly, phosphorylation at H3 tyrosine 41 was found to synergistically act together with H3K56ac to increase access to the peripheral DNA in the nucleosome68 (Figure 2d). Together, these studies highlight that the internal dynamics of the nucleosomes are modulated by a number of different factors, including histone variants, DNA sequence, and histone PTMs. This has direct effects on the DNA accessibility within chromatin, and thus impinges on transcription factor binding and dissociation kinetics with potential downstream consequences on gene expression control.

gave rise to small but measurable changes in the DNA conformation around the nucleosomes that could be explained by partial unwrapping. Subsequent investigations focused on a number of key acetylation sites in the globular domain of H3. H3K56 contacts the DNA phosphodiester backbone via a positioned water molecule close to the DNA entry site in the nucleosome structure.59 Acetylation of H3K56 was found to exhibit an important role in transcription and repair.60,61 To investigate a molecular function of this particular modification, Neumann et al. employed genetic code expansion methods to produce the modified histone recombinantly in bacterial cells.62 They used an engineered pair of an acetyl-lysyl-tRNA synthetase and a tRNA recognizing the amber stop codon63 to incorporate the acetyllysine at position 56 and reconstituted nucleosomes carrying fluorescent labels at defined DNA positions (Figure 2a). SmFRET studies of these nucleosomes demonstrated that the modification increased the probability for DNA unwrapping at the entry/exit site of the nucleosome up to 7-fold, whereas positions toward the dyad were not influenced.62 Together with small effects on chromatin remodeling, these studies showed how a single histone PTM can alter the dynamic behavior of nucleosomes. Expanding the set of core PTMs, Ottesen and colleagues devised a fully synthetic route to H3 by native chemical ligation of peptide fragments (NCL)64 allowing the incorporation of multiple PTMs at any site65 (Figure 2b). Together with the Poirier laboratory, they then investigated the function of three sets of acetylation marks, all positioned at important histone− DNA contacts: H3K56ac and H4(K77ac, K79ac) as well as H3(K115ac, K122ac).66 Employing force spectroscopy to probe nucleosome stability (Figure 2c), H3(K115ac, K122ac) were found to significantly weaken nucleosome integrity, resulting in chromatin disassembly upon DNA unwrapping. In contrast, both H3K56 and H4(K77ac, K79ac) did not affect overall stability but greatly increased transient DNA unwrapping events, resulting in the facilitation of TF access to

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INTERNAL MOTIONS IN CHROMATIN HIGHER ORDER STRUCTURE Arrays of nucleosomes can form higher order structures that critically affect chromatin function. At low ionic strength, chromatin arrays adopt extended bead-on-a-string conformations due to DNA charge repulsion. Under native solvent conditions, however, nucleosomes have an intrinsic propensity for self-interaction.36 Most importantly, interactions between the H4 tail and an acidic patch on histone H2A mediate nucleosome stacking and the formation of helical fibers with a diameter of about 30 nm (30 nm fibers).69 While 30 nm fibers have been observed in isolation,70 as well as in specific cell types,71 generally large-scale regular structure are not immediately visible in interphase nuclei.72−74 Rather, superresolution imaging of interphase nuclei revealed compact clusters of few nucleosomes connected by extended chromatin regions.75 Thus, 30 nm fibers may only exist over short distances in vivo, e.g. involving a few kilobases of DNA or tens of nucleosomes. Depending on the predominant internucleosomal contacts within chromatin fibers, two distinct topologies can be discriminated: The one-start, or solenoid, fiber model involves nearest-neighbor nucleosomal contacts. Conversely, two-start fibers are based on internucleosomal contacts between nucleosomes i and i + 2, to generate two stacks of nucleosomes D


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tweezers revealed behavior consistent with a one-start helix for the longer linkers (50 bp), whereas shorter linkers (20 bp) behaved more similarly to a two-start structure. The authors further quantified the interaction energy of nucleosomes as about 35 kJ/mol. Of note, this value was obtained for recombinant histones in the absence of any modifications and in the presence of divalent cations. Importantly, the low rigidity of the fibers led to the conclusion that chromatin fibers exhibit significant internal dynamics, resulting in large fluctuations in overall length. More details into the internal dynamics of oligonucleosomes were obtained using FRET spectroscopy. Poirier et al. prepared short arrays of three nucleosomes with carefully placed fluorescent dyes and measured internal dynamics by FRET-FCS82 (Figure 3b). These measurements revealed internal conformational fluctuations in the microsecond time regime. Accompanying stopped-flow measurements on compaction kinetics revealed slower dynamics on a seconds time scale related to nucleosome unstacking, thus demonstrating multiscale dynamics in chromatin arrays. Together with transient DNA unwrapping from nucleosomes, long-range fluctuation in fiber structure may thus be of great importance for transcription factor access to internal chromatin sites. Histone PTMs have been demonstrated to be intimately involved in chromatin structure regulation. Chromatin compaction experiments with truncated histone proteins identified the H4 tail to provide the major contacts stabilizing compact chromatin states.83 Surprisingly, acetylation of a single lysine residue in H4, K16 which was introduced by expressed protein ligation (EPL),84 proved sufficient to abolish chromatin fiber compaction, resulting in an expanded fiber of increased flexibility.85 In the following years, several other modifications that disrupt fiber folding were identified, including ubiquitylation of H2B86 and modification of the H4 tail with the small ubiquitin like modifier SUMO.87 To gain direct insight into the modulation of nucleosome−nucleosome contacts by histone PTMs, Lee et al. developed a smFRET-based interaction assay that allowed detection of stochastic interactions between surface-immobilized and diffusing nucleosomes in the presence of divalent cations58 (Figure 3c). The dynamics of such transient complexes provide information about nucleosome contacts within chromatin fibers. Employing this method, Lee et al. detected short-lived contacts between unmodified mononucleosomes in the 100 ms time range, as well as longlived interactions that persisted around 1 s. When the nucleosomes (in particular the H4 tail) were acetylated using Piccolo NuA4, the nucleosome−nucleosome interactions were significantly weakened as reflected in a 2-fold reduction of the interaction times and a pronounced 5-fold decrease in the fraction of long binding events. SUMOylation of H4 showed a similar effect, reducing the average lifetime of dinucleosomal interactions by about 20% and decreasing the formation frequency about 3-fold.87 The measured kinetics allowed determination of an interaction energy of about 23 kJ/mol between nucleosomes, corresponding to mid-micromolar dissociation constants. Taken together, chromatin fibers are highly dynamic and exhibit potentially competing topologies. Histone PTMs can critically modulate their structure and dynamics, thereby serving as gate keepers for DNA access on the nucleosome and chromatin fiber level.

which wind around a central axis and are connected by straight segments of linker DNA.76 Cross-linking studies could directly demonstrate i, i + 2 nucleosomal contacts.76 Furthermore, the X-ray structure of a tetranucleosome unit77 as well as recent cryo-electron microscopy structures of chromatin arrays with different linker lengths provided structural insight into the organization of a two start fiber model.78 Contrariwise, electron microscopy images of long chromatin fibers were more compatible with a solenoidal organization, in particular for long linker lengths and H1 incorporation.79 Furthermore, crosslinking studies coupled with modeling were compatible with heteromorphic chromatin structures, where solenoid and twostart structures coexist dependent on nucleosome positioning.80 Force spectroscopy is highly suitable to probe the energetics of chromatin fiber compaction. Cui and Bustamante used an optical tweezer to pull at a single chromatin fiber obtained from chicken erythrocytes.26 Interestingly, at physiological ionic strength and low force (5−6 pN) compaction and decompaction transitions could be monitored. The forces required to disrupt nucleosome−nucleosome interactions corresponded to interaction energies of 10 kJ/mol per nucleosome. These results thus demonstrated that under such conditions, chromatin fibers are dynamic structures exhibiting internal fluctuations and dynamic local opening and closing transitions. Expanding on those initial studies, Kruithof et al. employed defined reconstituted chromatin arrays of 25 nucleosomes with different linker DNA lengths, in the presence or absence of linker histones81 (Figure 3a). Force measurements by magnetic

Figure 3. Dynamics in chromatin higher order structure. (a) Stretching 25-mer arrays using magnetic tweezers reveals length fluctuations in arrays, and allows measurement of fiber stiffness as a function of DNA linker length, until complete fiber unstacking is reached.81 (b) Dynamic FRET measurements in a trinucleosomal system demonstrate multistate dynamics.82 Unstacking of neighboring nucleosomes appears as a two-step process, with the second step in the high ms time region (slow), whereas all the previous and subsequent reactions are in the μs time scale (fast). (c) A smFRET assay to measure dynamic nucleosome stacking demonstrates μM affinities and unstacking dynamics in the high ms to seconds regime.58,87 E


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Figure 4. Effector dynamics in chromatin. (a) Dynamic binding of HP1α to H3K9me3 carrying chromatin arrays,99 observed by single-molecule colocalization microscopy. (b) Chemical dimerization of HP1α by a structure guided, dual EPL reaction using a PxVxI peptide that interacts with the HP1α chromoshadow domain (CSD). (c) The HP1α residence time increases with both the local density of H3K9me3 marks and with multivalency in the protein. Multivalency also accelerates binding as seen in a decrease of the binding time. (d) DNA curtains are used to observe protein effector dynamics in immobilized chromatin arrays.106 (e) DNA curtains demonstrate that chromatin hinders the diffusion of the repair protein Msh2−Msh6, whereas the more flexible Mlh1−Pms1 can bypass nucleosomes.

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DYNAMIC READOUT OF HISTONE PTMS In addition to their intrinsic effects, arguably the most important role of histone PTMs stems from serving as an interaction platform for effector complexes. Chromatin effectors are recruited to genomic loci via the interaction of reader domains with specific PTM patterns.53,88 Chemical biology methods have proven to be instrumental in characterizing the affinity profiles of reader domains and their ability to synergistically function in recognizing combinatorial histone PTMs via multivalent interactions. Arrays of immobilized, synthetic histone peptides carrying defined modifications allowed probing of reader domains and detection of their targets.89,90 Conversely, protein arrays containing multiple recombinantly expressed reader domains enabled the identification of potential readers of a given modification.91,92 To probe interactions of full length constructs in a chromatin context, proteomic mass-spectroscopy-based approaches have proven highly successful,93 e.g., identifying multivalent binders of PTM patterns on histone H3.94,95 In addition, mass spectroscopy allowed the identification of readers of DNA methylation96 and hydroxymethylation marks.97 These methods do however not account for the dynamic competition of reader proteins for multiple nucleosomes containing different combinations of PTMs, as encountered in the nucleus. To circumvent these shortcomings, and to develop a highly multiplexed assay to obtain the dynamic interaction profile for a given chromatin reader complex, Nguyen et al. employed EPL to produce a semisynthetic library of specifically modified nucleosomes.98 The modification state of each nucleosome was then encoded in the wrapped nucleosomal DNA and the library, encompassing a large number of different chromatin states, was pooled. Nucleosomes interacting with chromatin effectors, such as the KAT p300, were enriched and identified by next-generation DNA sequencing, thereby revealing the chromatin affinity profile of the factor under investigation. While this method lacks single molecule resolution, it is highly sensitive due to signal amplification in DNA sequencing and it can account for dynamic binding across differently modified chromatin states. What, however, are the mechanistic consequences of multivalent chromatin interactions of an effector protein within the context of a chromatin fiber? Within the nonequilibrium

environment of the cell, interaction kinetics, i.e. binding rates and residence times, determine the downstream biological effect of a chromatin-effector interaction. Kilic et al. developed a single-molecule approach to observe transient interactions of chromatin effectors, for example, with HP1α, with modified chromatin arrays in a fully reconstituted system99 (Figure 4a). HP1α is a major factor in transcriptionally silent heterochromatin and was found to be highly dynamic on a molecular level in cells.12 HP1α contains a chromodomain (CD) that specifically binds to H3K9me3, the canonical heterochromatic histone PTM, with micromolar affinity.53 Furthermore, it exists as a dimer and is thus able to engage multiple H3K9 methylated nucleosomes in a multivalent fashion, which has been suggested to result in chromatin compaction by nucleosome crossbridging.100 Nuclear magnetic resonance measurements showed that HP1β, a close homologue, interacting with single nucleosomes forms highly dynamic complexes.101 Expanding on these studies with the aim to explore the dynamic behavior of HP1α in a chromatin fiber context, Fierz and colleagues reconstituted defined H3K9me3 carrying nucleosome arrays, which were immobilized in microfluidic channels. This allowed the direct observation of the interaction kinetics of fluorescently labeled HP1α with single chromatin arrays over extended time by smTIRF microscopy. The authors determined average residence times of 250 ms for a majority of HP1α proteins and a longer-lived population that remained attached for seconds. Heterogeneity in the observed kinetics between different chromatin arrays potentially arise from distinct conformational states. Subsequent systematic changes of the chromatin H3K9me3 density and chemical modulation of the HP1α oligomerization state (Figure 4b) allowed probing of the mechanism of HP1α−chromatin recognition. These experiments revealed that, first, HP1α binding dynamics critically depend on the density of H3K9me3 marks on the chromatin fiber (Figure 4c). High PTM density allows multivalent binding, and it also enables transiently dissociated reader proteins to rapidly reassociate at a neighboring site. Second, multivalent binding of HP1α results in both an increase in residence time as well as, intriguingly, an increase in the association rate. In summary, effector−chromatin interactions are highly dynamic and depend on the high local density of a given PTM pattern. The proteins are thus kinetically captured F


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Figure 5. Effector dynamics on modified chromatin in living cells. (a) Target search of the TF Sox2:116 Single-molecule imaging of Sox2 (and Oct4), labeled with tetramethylrhodamine via a Halo-tag,121 imaged in ES cells revealing the time spent diffusing in the nucleoplasm (τ3D) between binding events, as well as residence times (τres) at nonspecific and specific chromatin sites. Alteration of the chromatin state by TSA results in an increase of available target sites, a decrease in the diffusion time, as well as an increase in the residence times. (b) FabLEM: A Fab, produced from a histone PTM-specific monoclonal antibody by digestion followed by fluorescent labeling, stains histone PTMs after loading into a cell.119 (c) Scheme of FabLEM-based imaging of transcription:120 H3K27 is acetylated before activation by glucocorticoid receptor (GR, labeled by GFP), followed by RNA polymerase 2 (RNAP2) recruitment and elongation.

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OBSERVING CHROMATIN INTERACTIONS IN THE CELL At the core of chromatin regulatory processes lies the recruitment of protein effectors to a particular genomic locus or chromatin state within the living cell. This poses a formidable biological problem, as the nuclear environment is crowded; specific binding sites are potentially rare and surrounded by large stretches of nonspecific chromatin, requiring a highly efficient target search process. Furthermore, when bound, the biological output of a chromatin effector is coupled to its local residence time. Thus, in vivo determination of both the association and dissociation kinetics of TFs, epigenetic effectors, and other chromatin associated proteins are of key importance for a deeper understanding of their function. In recent years, multiple methods have been developed to monitor epigenetic and gene regulatory processes with high time resolution on the single cell or even singlemolecule level. Most of the research in this area has been performed to understand the dynamic behavior of TFs, to elucidate the mechanisms for finding their genomic targets, and to characterize their ability to regulate gene expression. It has long been recognized that TF residence times are linked to their ability to direct transcriptional outcomes.108 Even at similar average occupancy, gene promoters with stably bound TFs exhibit higher gene expression as compared to promoters with fast exchanging TFs.109,110 These results showcase that TF dynamics are intricately related to their activity within a chromatin environment, as efficient gene activation, including complex processes such as chromatin remolding, require a complex that is stable over an extended amount of time. Can such localized complex assembly between transcription factors within a chromatin environment be monitored directly? Recent developments in observing TF dynamics in living cells

in a given chromatin region, thereby enacting a biological function, while allowing the possibility of a rapid change in chromatin state as required for mitosis102 or in DNA damage repair.103

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VISUALIZING PROTEIN DIFFUSION ON CHROMATIN

Epigenetic effectors as well as TFs or repair proteins are constantly scanning the genome for suitable binding sites. Nanofabricated DNA curtains, established by Greene and colleagues, provide a highly versatile platform to observe chromatin processes over the long range104 (Figure 4d). DNA curtains permit chromatin assembly, and such methods have been employed to investigate the energetic bias for nucleosome positioning by local DNA sequence.105 Similarly, chromatinized DNA curtains enabled Gorman et al. to investigate the diffusional search process of factors involved in DNA damage repair.106 They investigated two mismatch repair (MMR) factors from yeast, Msh2−Msh6 and Mlh1−Pms1, for their ability to traverse nucleosomes within chromatin. Single molecule observations revealed that Msh2−Msh6 moves along DNA by a sliding mechanism107 that is inhibited by the presence of chromatin. The Mlh1−Pms1 heterodimer, in contrast, forms a ring-like structure by means of long linker arms, which enables more flexibility and almost unhindered passage of nucleosomes (Figure 4e). It is thus clear that target search mechanisms of TFs or repair proteins based on DNA sliding will be greatly influenced by nucleosomes and chromatin fibers. In summary, future studies combining chemically defined chromatin and single-molecule methods will be instrumental for a greater understanding of the behavior of epigenetic effectors, TFs, or other nuclear proteins functioning on their native substrate. G


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pattern, histone variants, and combinations of PTMs. Chemical biology approaches to synthesize and reconstitute defined chromatin states, together with high-powered single-molecule imaging, allow revealing the intricate details of chromatin regulation in real time and with molecular resolution. Here, I have outlined progress that has been made in recent years in studying chromatin systems of high complexity. First, progress in the production of chemically defined chromatin in almost any conceivable modification state and architecture allows for detailed investigations of its function. Second, biophysical methods of increasing sophistication enable the exploration of the energetics of nucleosomes or chromatin fibers and the intrinsic influence of individual or combinatorial PTMs. Conversely, single-molecule approaches are progressively amenable to supramolecular systems previously accessible only to classical biochemical approaches. Finally, single-particle tracking approaches allow direct relation of in vitro experiments with chemically defined components to observation in cells. A major challenge for the near future is to further integrate biochemistry and chemical biology with single-molecule imaging with the goal of studying entire chromatin signaling pathways with defined components. Quantitative results from such experiments can then be used for a systems description of chromatin regulatory processes with predictive power of signaling outcomes in living cells and organisms.

by a number of laboratories, including the use of sparse labeling or photoactivation protocols, as well as employing long exposure times or light sheet illumination, paved the way for more detailed investigations of such processes.111−114 In ES cells, the key pluripotency factors Sox2 and Oct4 search the chromatin independently for binding sites but form complexes on enhancer regions, enhanceosomes, thereby modulating gene expression.115 Chen et al. employed single-molecule imaging in vivo and in vitro to visualize target search and enhanceosome assembly.116 Using long exposure times, which result in blurring out diffusing Sox2 molecules while allowing detection of chromatin bound molecules, the authors could discriminate between nonspecific DNA binding events with residence times below 1 s and more stably bound specific binding events with residence times in the tens of seconds. Single-molecule tracking by multifocus microscopy,112 conversely, revealed the search kinetics between binding events. Sox2 was found to serve as a lead factor, binding first and stabilizing Oct4 binding, demonstrating hierarchical assembly of the enhanceosome complex. Furthermore, the chromatin state directly influenced search kinetics of the TFs: Treatment of the cells with Trichostatin A (TSA), an inhibitor of HDACs that raises global histone acetylation,117 decreased the search time for specific sites by opening chromatin, thereby exposing a larger number of specific binding sites (Figure 5a). A similar effect was observed for 5-azacytidine, which reduces DNA methylation,118 thereby directly highlighting the relationship between chromatin state, structure, and TF transactions. The dynamics of histone modifications, themselves, are not visible in such tracking experiments. Directly visualizing PTM turnover in single cells allows real-time detection of chromatin state changes. In particular, the ability to microinject fluorescently labeled and highly specific antibodies, or fragment antigen-binding (Fab fragment) regions, has been used to monitor PTM dynamics119 (Figure 5b). Combining Fab-based live endogenous modification labeling (FabLEM)119 with dynamic single-cell imaging, Stasevich et al. could directly observe the transcription process and associated changes in the chromatin state with seconds time resolution.120 The authors correlated the dynamics of one acetyl mark, H3K27ac, with RNA polymerase 2 (RNAP2) binding and phosphorylation within a reporter gene array. Local levels of H3K27ac are initially high and then drop during transcription onset (Figure 5c). Interestingly, cell-to-cell differences in initial H3K27ac levels could be related to the transcription process: A localized increase in H3K27 acetylation directly correlated with efficient transcription. Based on kinetic modeling, the authors suggested that locus specific enhanced acetylation might result in more efficient TF access, as well as a stimulation of RNAP2 promoter escape. Taken together, local interplay between PTM writer and eraser enzymes modulate chromatin dynamics and thus directly impinge upon gene expression. The stochastic nature of this process results in large temporal variability, as well as between different individual cells or for individual genes. Thus, future studies are required to reveal how feedback regulatory processes allow the generation of stable cell populations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: beat.fierz@epfl.ch. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I thank S. Kilic for discussions and comments on the manuscript. I gratefully acknowledge the Sandoz Family Foundation, the Swiss National Science Foundation (grant 31003A_149789), the NCCR Chemical Biology and EPFL for financial support.

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CONCLUSIONS Chromatin regulation involves highly complex biochemical processes that rely on the interplay between a large number of protein factors. Chromatin itself is a very dynamic nucleoprotein complex with properties dependent on the local DNA sequence, the positioning of nucleosomes, DNA methylation H

KEYWORDS: Chromatin: nucleoprotein complex between histone proteins and DNA, as well as multiple nonhistone proteins and RNA molecules, organizing the chromosomes in eukaryotic cells Chromatin effectors: proteins that interact with the chromatin fiber, often via histone modification specific reader domains, thereby modulating chromatin properties, rewriting histone PTMs, or altering gene expression Chromatin state: the coexisting DNA and histone modification patterns, together with the present histone variants, nucleosome positions, and bound chromatinassociated proteins at a given genomic locus Total internal reflection fluorescence microscopy: microscopy technique, where an excitation laser beam is totally reflected at the coverglass−specimen interface, resulting in the selective illumination of a thin specimen layer up to around 200 nm from the coverglass surface Single-molecule fluorescence resonance energy transfer: distance-dependent energy transfer between a donor and acceptor dye, allowing dynamic measurement of the dye separation based on the ratio of observed fluorescence emission from donor and acceptor dye in single molecules DOI: 10.1021/acschembio.5b00832 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Magnetic tweezers: force spectroscopy method that allows the manipulation of single molecules attached to a magnetic particle by exerting force through an applied magnetic field Optical tweezers: force spectroscopy method where force is exerted on a single molecule that is attached to a dielectric particle trapped in a highly focused laser beam Genetic code expansion: method to introduce non-natural or modified amino acids into proteins employing an engineered pair of a tRNA (tRNA) recognizing the rare “amber” stop codon and a corresponding aminoacyl-tRNA synthetase that loads the non-native amino acid on the tRNA Expressed protein ligation: method to introduce non-natural or modified amino acids into proteins by linking a synthetic peptide to an expressed protein fragment via the formation of a peptide bond between a C-terminal thioester and an Nterminal cysteine residue

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Dynamic Chromatin Regulation from a Single Molecule Perspective

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