Methods 70 (2014) 75–76
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Editorial
Putting numbers on chromatin and its interacting partners
Chromatin is a difficult substrate to study: it is large, difficult to manipulate, extremely heterogenous, and dynamic. Non-specific interactions abound, and its very preparation can significantly disturb its composition and structure. None of these attributes are particularly conducive to quantitative analysis. However, if we want to understand how access to the genome is regulated, we must understand its organization, its multiple interactions, and the forces that hold it together. Methods to do just that are being developed at an ever increasing rate, and techniques are becoming more and more complex and complicated. Having been active in the field of chromatin structure and function for over two decades, I am amazed by the things we can do now. Just to name a few, we can now map nucleosome positions to near base pair resolution, we can derive thermodynamic binding constants from chromatin immuno-precipitation data, and we can quantify the dynamics of post-translational modifications, all within the context of the cell. Developments in the in vitro analysis of chromatin and chromatin-interacting proteins are no less impressive, and a series of papers describes recent advances in synthetizing non-hydrolyzable ubiquitinated histones, in quantifying post-translational modifications, and in using a variety of approaches to study nucleosome stability and the structure of the chromatin fiber. For new methods to be accessible to a general audience, the experimental procedures must be published at a level of detail that is often not supported by the increasingly terse and data-dense publication format that prevails today. Many high-ranking journals have delegated the Materials and Methods section to a few paragraphs in the Supplementary data (where they are rarely peer reviewed), or the authors refer to a maze of references in hardto-find journals. This is particularly worrisome as the methods are becoming increasingly complex, and the devil, now more than ever, is buried in the details. More often than not, attempts to reproduce a method based on published research papers are doomed because key details are missing. Dissemination of method is thus unnecessarily delayed. Detailed, step-by-step descriptions provided by the leaders in the field who pioneered these approaches are extremely valuable resources, and they provide a great service to the scientific community. This volume combines nine such contributions that will be useful resources for years to come and will contribute to putting these methods to wide use. A common theme that runs through all contributions is a shift from descriptive to quantitative approaches in analyzing the various aspects of chromatin. In the first contribution, Stasevitch and colleagues describe an antibody-based approach (Fab-based Live Endogenous Modification Labeling, or FabLEM) to simultaneously track specific histone http://dx.doi.org/10.1016/j.ymeth.2014.12.006 1046-2023/Ó 2014 Published by Elsevier Inc.
modifications together with active RNA polymerase in a single cell [1]. This can even be done in subsequent generations. This method will be uniquely useful to dissect the interplay between histone modifications and the transcription machinery, and to test how modifications correlate over generations. The next two contributions describe advances in the widely used chromatin immunoprecipitation (ChIP) technique. In the contribution by Ciborowski [2], ChIP is applied to human monocytederived macrophages to show that differences exist among various primary cell lines and cells derived from volunteers. One major drawback of ChIP assays is that they essentially provide a yes/no answer on chromatin-interacting proteins. Information on the particular strength or off-rate of any given interaction is lost. In a breakthrough by the Auble and Bekiranov labs, commonly used ChIP approaches are taken to another level by including the time dimension. In the Crosslinking kinetics assay (CLK assay, aptly pronounced ‘Clock assay’), the time-dependence of formaldehyde crosslinking is used to extract on- and off-rates in vivo [3]. When widely applied to the myriad of known (and as yet unknown) chromatin interactions, this approach will provide a detailed (and likely drastically different) picture of many chromatin-related events. Every DNA binding/processing protein must contend with the fact that the DNA is tightly wound around the surface of the histone octamer. It is thus essential to understand the dynamics and accessibility of nucleosomal DNA. Poirier and colleagues have developed an elegant single-molecule FRET assay that compares the binding of DNA binding proteins with free DNA and with nucleosomes and nucleosomal arrays [4]. Incidentally, it also gives information on how tightly nucleosomal DNA is bound to the histone octamer core. Kurumizaka describes a versatile method for evaluating nucleosome stability, and this approach will be uniquely suited to probe the effects of DNA sequence, histone variants and histone post-translational modifications on nucleosome stability and thus DNA accessibility [5]. Histones are acetylated on many different sites in a transient manner, and this dynamic post-translational modification has essential signaling functions. Andrews and colleagues present a novel approach to quantify histone modifications using a modified mass spectrometry protocol [6]. This approach can also be used to understand the specificity and efficiency of the enzymes that are responsible for adding these modifications, the histone acetyl transferases. Arguably, ubiquitination is perhaps the strangest of all the histone post-translational modifications. Due to its size, the effects of ubiquitin conjugation on nucleosome and chromatin structure are not easily predictable, nor is it known which protein factors
76
Editorial / Methods 70 (2014) 75–76
specifically recognize nucleosomes and histones bearing this modification. Yao and colleagues describe a beautiful approach to preparing non-hydrolyzable ubiquitinated forms of histones [7]. Ubiquitin can be attached to any site, with 100% efficiency, thus enabling the identification of ‘readers’ of this intriguing modification. The last two contributions describe approaches that are perhaps a bit less mainstream, but nonetheless very powerful methods for obtaining otherwise hard-to-come-by structural information on protein complexes and on the chromatin fiber. In the contribution by Owen-Hughes and colleagues, a combination of site-specific spin labeling and Pulsed Electron Double Resonance (PELDOR) is applied to a histone chaperone – histone pair [8]. Together with other constraints, the distance measurements obtained from PELDOR can be used to arrive at a low-resolution model of the overall architecture of the complex. The structure of the chromatin fiber remains the ‘holy grail’ of structural biology, because it is particularly inaccessible to many of the standard structural approaches. The classical model of the hierarchical organization of 10 nm (‘beads on a string’) chromatin into an ordered 30 nm fiber has recently been challenged. Maeshima and colleagues use Small Angle X-ray Scattering (SAXS) to determine that no structural features larger than 11 nm are found in interphase chromatin or mitotic chromosomes [9]. This
approach further cements the relatively new notion that chromatin essentially consists of irregularly folded nucleosome fibers. Combined with ongoing efforts to map the three-dimensional interactions of chromatin in the nucleus, this technique will likely contribute significantly to our understanding of the structure of the human chromosome. References [1] T.J. Stasevich, Y. Sato, N. Nozaki, H. Kimura, Methods 70 (2–3) (2014) 77–88. [2] J. Wooden, P. Ciborowski, Methods 70 (2–3) (2014) 89–96. [3] R. Viswanathan, E.A. Hoffman, S.J. Shetty, S. Bekiranov, D.T. Auble, Methods 70 (2–3) (2014) 97–107. [4] Y. Luo, J.A. North, M.G. Poirier, Methods 70 (2–3) (2014) 108–118. [5] H. Taguchi, N. Horikoshi, Y. Arimura, H. Kurumizaka, Methods 70 (2–3) (2014) 119–126. [6] Y.-M. Kuo, R.A. Henry, A.J. Andrews, Methods 70 (2–3) (2014) 127–133. [7] L. Long, M. Furgason, T. Yao, Methods 70 (2–3) (2014) 134–138. [8] C.M. Hammond, T. Owen-Hughes, D.G. Norman, Methods 70 (2–3) (2014) 139– 153. [9] K. Maeshima, R. Imai, T. Hikima, Y. Joti, Methods 70 (2–3) (2014) 154–161.
Karolin Luger HHMI and Department of Biochemistry and Molecular Biology, Colorado State University, United States
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Editorial
Putting numbers on chromatin and its interacting partners
Chromatin is a difficult substrate to study: it is large, difficult to manipulate, extremely heterogenous, and dynamic. Non-specific interactions abound, and its very preparation can significantly disturb its composition and structure. None of these attributes are particularly conducive to quantitative analysis. However, if we want to understand how access to the genome is regulated, we must understand its organization, its multiple interactions, and the forces that hold it together. Methods to do just that are being developed at an ever increasing rate, and techniques are becoming more and more complex and complicated. Having been active in the field of chromatin structure and function for over two decades, I am amazed by the things we can do now. Just to name a few, we can now map nucleosome positions to near base pair resolution, we can derive thermodynamic binding constants from chromatin immuno-precipitation data, and we can quantify the dynamics of post-translational modifications, all within the context of the cell. Developments in the in vitro analysis of chromatin and chromatin-interacting proteins are no less impressive, and a series of papers describes recent advances in synthetizing non-hydrolyzable ubiquitinated histones, in quantifying post-translational modifications, and in using a variety of approaches to study nucleosome stability and the structure of the chromatin fiber. For new methods to be accessible to a general audience, the experimental procedures must be published at a level of detail that is often not supported by the increasingly terse and data-dense publication format that prevails today. Many high-ranking journals have delegated the Materials and Methods section to a few paragraphs in the Supplementary data (where they are rarely peer reviewed), or the authors refer to a maze of references in hardto-find journals. This is particularly worrisome as the methods are becoming increasingly complex, and the devil, now more than ever, is buried in the details. More often than not, attempts to reproduce a method based on published research papers are doomed because key details are missing. Dissemination of method is thus unnecessarily delayed. Detailed, step-by-step descriptions provided by the leaders in the field who pioneered these approaches are extremely valuable resources, and they provide a great service to the scientific community. This volume combines nine such contributions that will be useful resources for years to come and will contribute to putting these methods to wide use. A common theme that runs through all contributions is a shift from descriptive to quantitative approaches in analyzing the various aspects of chromatin. In the first contribution, Stasevitch and colleagues describe an antibody-based approach (Fab-based Live Endogenous Modification Labeling, or FabLEM) to simultaneously track specific histone http://dx.doi.org/10.1016/j.ymeth.2014.12.006 1046-2023/Ó 2014 Published by Elsevier Inc.
modifications together with active RNA polymerase in a single cell [1]. This can even be done in subsequent generations. This method will be uniquely useful to dissect the interplay between histone modifications and the transcription machinery, and to test how modifications correlate over generations. The next two contributions describe advances in the widely used chromatin immunoprecipitation (ChIP) technique. In the contribution by Ciborowski [2], ChIP is applied to human monocytederived macrophages to show that differences exist among various primary cell lines and cells derived from volunteers. One major drawback of ChIP assays is that they essentially provide a yes/no answer on chromatin-interacting proteins. Information on the particular strength or off-rate of any given interaction is lost. In a breakthrough by the Auble and Bekiranov labs, commonly used ChIP approaches are taken to another level by including the time dimension. In the Crosslinking kinetics assay (CLK assay, aptly pronounced ‘Clock assay’), the time-dependence of formaldehyde crosslinking is used to extract on- and off-rates in vivo [3]. When widely applied to the myriad of known (and as yet unknown) chromatin interactions, this approach will provide a detailed (and likely drastically different) picture of many chromatin-related events. Every DNA binding/processing protein must contend with the fact that the DNA is tightly wound around the surface of the histone octamer. It is thus essential to understand the dynamics and accessibility of nucleosomal DNA. Poirier and colleagues have developed an elegant single-molecule FRET assay that compares the binding of DNA binding proteins with free DNA and with nucleosomes and nucleosomal arrays [4]. Incidentally, it also gives information on how tightly nucleosomal DNA is bound to the histone octamer core. Kurumizaka describes a versatile method for evaluating nucleosome stability, and this approach will be uniquely suited to probe the effects of DNA sequence, histone variants and histone post-translational modifications on nucleosome stability and thus DNA accessibility [5]. Histones are acetylated on many different sites in a transient manner, and this dynamic post-translational modification has essential signaling functions. Andrews and colleagues present a novel approach to quantify histone modifications using a modified mass spectrometry protocol [6]. This approach can also be used to understand the specificity and efficiency of the enzymes that are responsible for adding these modifications, the histone acetyl transferases. Arguably, ubiquitination is perhaps the strangest of all the histone post-translational modifications. Due to its size, the effects of ubiquitin conjugation on nucleosome and chromatin structure are not easily predictable, nor is it known which protein factors
76
Editorial / Methods 70 (2014) 75–76
specifically recognize nucleosomes and histones bearing this modification. Yao and colleagues describe a beautiful approach to preparing non-hydrolyzable ubiquitinated forms of histones [7]. Ubiquitin can be attached to any site, with 100% efficiency, thus enabling the identification of ‘readers’ of this intriguing modification. The last two contributions describe approaches that are perhaps a bit less mainstream, but nonetheless very powerful methods for obtaining otherwise hard-to-come-by structural information on protein complexes and on the chromatin fiber. In the contribution by Owen-Hughes and colleagues, a combination of site-specific spin labeling and Pulsed Electron Double Resonance (PELDOR) is applied to a histone chaperone – histone pair [8]. Together with other constraints, the distance measurements obtained from PELDOR can be used to arrive at a low-resolution model of the overall architecture of the complex. The structure of the chromatin fiber remains the ‘holy grail’ of structural biology, because it is particularly inaccessible to many of the standard structural approaches. The classical model of the hierarchical organization of 10 nm (‘beads on a string’) chromatin into an ordered 30 nm fiber has recently been challenged. Maeshima and colleagues use Small Angle X-ray Scattering (SAXS) to determine that no structural features larger than 11 nm are found in interphase chromatin or mitotic chromosomes [9]. This
approach further cements the relatively new notion that chromatin essentially consists of irregularly folded nucleosome fibers. Combined with ongoing efforts to map the three-dimensional interactions of chromatin in the nucleus, this technique will likely contribute significantly to our understanding of the structure of the human chromosome. References [1] T.J. Stasevich, Y. Sato, N. Nozaki, H. Kimura, Methods 70 (2–3) (2014) 77–88. [2] J. Wooden, P. Ciborowski, Methods 70 (2–3) (2014) 89–96. [3] R. Viswanathan, E.A. Hoffman, S.J. Shetty, S. Bekiranov, D.T. Auble, Methods 70 (2–3) (2014) 97–107. [4] Y. Luo, J.A. North, M.G. Poirier, Methods 70 (2–3) (2014) 108–118. [5] H. Taguchi, N. Horikoshi, Y. Arimura, H. Kurumizaka, Methods 70 (2–3) (2014) 119–126. [6] Y.-M. Kuo, R.A. Henry, A.J. Andrews, Methods 70 (2–3) (2014) 127–133. [7] L. Long, M. Furgason, T. Yao, Methods 70 (2–3) (2014) 134–138. [8] C.M. Hammond, T. Owen-Hughes, D.G. Norman, Methods 70 (2–3) (2014) 139– 153. [9] K. Maeshima, R. Imai, T. Hikima, Y. Joti, Methods 70 (2–3) (2014) 154–161.
Karolin Luger HHMI and Department of Biochemistry and Molecular Biology, Colorado State University, United States
