chromatin remodeling: a collaborative effort

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14 VOLUME 21 NUMBER 1 JANUARY 2014 NATURE STRUCTURAL & MOLECULAR BIOLOGY evidence that mitochondrial-derived cofac- tors, such as FeS clusters, play crucial roles in the metabolism and health of eukaryotic cells. With the structure of the catalytic subunit of polymerase ε accounted for, its regulatory subunits, Dpb2, Dpb3 and Dpb4, remain to be described. We will be eagerly awaiting the structure of the holoenzyme. ACKNOWLEDGMENTS Work in the authors’ laboratory was supported by US National Institutes of Health grant R01 CA052040. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Johansson, E. & Macneill, S.A. Trends Biochem. Sci. 35, 339–347 (2010). 2. Miyabe, I., Kunkel, T.A. & Carr, A.M. PLoS Genet. 7, e1002407 (2011). 3. Hogg, M. et al. Nat. Struct. Mol. Biol. 21, 49–55 (2014). 4. Swan, M.K., Johnson, R.E., Prakash, L., Prakash, S. & Aggarwal, A.K. Nat. Struct. Mol. Biol. 16, 979–986 (2009). 5. Baranovskiy, A.G. et al. Cell Cycle 7, 3026–3036 (2008). 6. Steitz, T.A. J. Biol. Chem. 274, 17395–17398 (1999). 7. Church, D.N. et al. Hum. Mol. Genet. 22, 2820–2828 (2013). 8. Perera, R.L. et al. eLife 2, e00482 (2013). 9. Pavlov, Y.I. et al. Curr. Biol. 16, 202–207 (2006). 10. Jain, R. et al. J. Mol. Biol. doi:10.1016/ j.jmb.2013.10.015 (19 October 2013). 11. Netz, D.J. et al. Nat. Chem. Biol. 8, 125–132 (2012). 12. Wolski, S.C., Kuper, J. & Kisker, C. Biol. Chem. 391, 761–765 (2010). 13. Li, V., Hogg, M. & Reha-Krantz, L.J. J. Mol. Biol. 400, 295–308 (2010). 14. Hogg, M., Aller, P., Konigsberg, W., Wallace, S.S. & Doublié, S. J. Biol. Chem. 282, 1432–1444 (2007). implicated in DNA double-strand-break repair, in which they perform overlapping and distinct functions 13 . Many different remodelers are also targeted to replication sites and have roles during or after the DNA replication process 14 . The centromere is another well-studied chromatin struc- ture in which many remodeling factors act together 15–20 . Mammalian genomes are orders of magni- tude larger and more complex than are yeast genomes. Huge ‘gene deserts’ often separate protein-coding genes that themselves may be spread over tens to hundreds of kilobases of DNA and often contain many and large introns. Several studies indicate that in mam- malian cells members of distinct classes of nucleosome-remodeling factors act on a specific gene or locus, often in a successive manner, sometimes with mutual dependence and at other times in opposition 21–23 . Major questions, therefore, are why are there so many different nucleosome-remodeling factors expressed in the same cell, and to what extent do these activities collaborate or control each other to shape the dynamic genome? In this issue, a study 24 from the Hager labo- ratory addresses these questions by analyzing where three nucleosome-remodeling ATPases bind genome wide and how they cooperate to shape chromatin in a mouse mammary epi- thelial cell line. The researchers mapped the distribution of Brg1, Snf2h and Chd4 ATPases, which are at the core of multiple complexes (Fig. 1). Morris et al. 24 used chromatin The existence of such a large number of factors is explained, in part, by tissue- specific expression and function of some chromatin-remodeling complexes 4 . However, numerous remodeling factors are expressed simultaneously in the same cell type, and this prompts questions about their spe- cific roles. Many of them interact with and discriminate between the various post- translationally modified histones. This can be seen as an endpoint of distinct signaling cascades acting on chromatin. Furthermore, some of these enzymes show specialized nucleosome-remodeling activities. For instance, in budding yeast the SWR1 and INO80 complexes catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosomes over the transcription start sites of genes 6 . However, many nucleosome-remodeling factors exhibit similar functions when assayed in vitro 7 . And, whereas some genes encoding nucleosome-remodeling factors are essential or required for robust viability in yeast, oth- ers can be deleted singly, without any major defect in viability, thus indicating functional redundancy 8 . Studies in yeast illustrate how various nucleosome-remodeling factors have overlapping and complementary functions to shape chromatin over and around genes, for example, in regulating the progression of RNA polymerase II 9–12 . Evidence for col- laboration between different remodelers is not restricted to gene regulation. Almost all classes of remodeler ATPases have been The packaging of eukaryotic genomes into nucleosome-based chromatin necessitates mechanisms that regulate the access to DNA of, for example, the transcription machinery. The past ~20 years have revealed a plethora of different enzymes that modify chromatin, including chromatin- or nucleosome- remodeling enzymes that use energy gained from ATP hydrolysis to alter nucleosome struc- ture. Saccharomyces cerevisiae alone encodes eight factors with demonstrated nucleosome- remodeling activity similar to that of SWI2/ SNF2 ATPase 1 . The picture is complicated further by the fact that a single SWI2/SNF2- like motor protein often forms several distinct complexes by stably interacting with other proteins 2,3 (Fig. 1). These accessory subunits are involved in regulating the catalytic activity of the complexes; some of them bind histone- modifying enzymes or modified histones themselves, or are involved in recruitment by sequence-specific transcriptional regula- tors. In higher eukaryotes, there are many more nucleosome remodelers and distinct remodeling complexes formed around the same core ATPase 2–4 . Deletion of many of them leads to early embryonic lethality, and mutations in genes encoding these factors are implicated in several genetic diseases, most notably cancer 2–5 . Chromatin remodeling: a collaborative effort Patrick D Varga-Weisz Enzymes that alter nucleosome structure or position are at the very center of gene and genome regulation, and understanding how, and to what extent, these diverse activities collaborate and control each other to shape the genome for dynamic regulation is a major challenge. A new study provides an important step in this direction by illustrating the cooperative nature of ATP-dependent chromatin-remodeling systems in mammalian cells. Patrick D. Varga-Weisz is at the Nuclear Dynamics Programme, Babraham Institute, Cambridge, UK. e-mail: [email protected] NEWS AND VIEWS npg © 2014 Nature America, Inc. All rights reserved.

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Page 1: Chromatin remodeling: a collaborative effort

14 volume 21 number 1 JAnuArY 2014 nature structural & molecular biology

evidence that mitochondrial-derived cofac-tors, such as FeS clusters, play crucial roles in the metabolism and health of eukaryotic cells. With the structure of the catalytic subunit of polymerase ε accounted for, its regulatory subunits, Dpb2, Dpb3 and Dpb4, remain to be described. We will be eagerly awaiting the structure of the holoenzyme.

ACKNOWLEDGMENTSWork in the authors’ laboratory was supported by US National Institutes of Health grant R01 CA052040.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

1. Johansson, E. & Macneill, S.A. Trends Biochem. Sci. 35, 339–347 (2010).

2. Miyabe, I., Kunkel, T.A. & Carr, A.M. PLoS Genet. 7, e1002407 (2011).

3. Hogg, M. et al. Nat. Struct. Mol. Biol. 21, 49–55 (2014).4. Swan, M.K., Johnson, R.E., Prakash, L., Prakash, S. &

Aggarwal, A.K. Nat. Struct. Mol. Biol. 16, 979–986 (2009).

5. Baranovskiy, A.G. et al. Cell Cycle 7, 3026–3036 (2008).

6. Steitz, T.A. J. Biol. Chem. 274, 17395–17398 (1999).

7. Church, D.N. et al. Hum. Mol. Genet. 22, 2820–2828 (2013).

8. Perera, R.L. et al. eLife 2, e00482 (2013).9. Pavlov, Y.I. et al. Curr. Biol. 16, 202–207 (2006).10. Jain, R. et al. J. Mol. Biol. doi:10.1016/

j.jmb.2013.10.015 (19 October 2013).11. Netz, D.J. et al. Nat. Chem. Biol. 8, 125–132

(2012).12. Wolski, S.C., Kuper, J. & Kisker, C. Biol. Chem. 391,

761–765 (2010).13. Li, V., Hogg, M. & Reha-Krantz, L.J. J. Mol. Biol. 400,

295–308 (2010).14. Hogg, M., Aller, P., Konigsberg, W., Wallace, S.S.

& Doublié, S. J. Biol. Chem. 282, 1432–1444 (2007).

implicated in DNA double-strand-break repair, in which they perform overlapping and distinct functions13. Many different remodelers are also targeted to replication sites and have roles during or after the DNA replication process14. The centromere is another well-studied chromatin struc-ture in which many remodeling factors act together15–20.

Mammalian genomes are orders of magni-tude larger and more complex than are yeast genomes. Huge ‘gene deserts’ often separate protein-coding genes that themselves may be spread over tens to hundreds of kilobases of DNA and often contain many and large introns. Several studies indicate that in mam-malian cells members of distinct classes of nucleosome-remodeling factors act on a specific gene or locus, often in a successive manner, sometimes with mutual dependence and at other times in opposition21–23. Major questions, therefore, are why are there so many different nucleosome-remodeling factors expressed in the same cell, and to what extent do these activities collaborate or control each other to shape the dynamic genome?

In this issue, a study24 from the Hager labo-ratory addresses these questions by analyzing where three nucleosome-remodeling ATPases bind genome wide and how they cooperate to shape chromatin in a mouse mammary epi-thelial cell line. The researchers mapped the distribution of Brg1, Snf2h and Chd4 ATPases, which are at the core of multiple complexes (Fig. 1). Morris et al.24 used chromatin

The existence of such a large number of factors is explained, in part, by tissue-specific expression and function of some chromatin-remodeling complexes4. However, numerous remodeling factors are expressed simultaneously in the same cell type, and this prompts questions about their spe-cific roles. Many of them interact with and discriminate between the various post- translationally modified histones. This can be seen as an endpoint of distinct signaling cascades acting on chromatin. Furthermore, some of these enzymes show specialized nucleosome-remodeling activities. For instance, in budding yeast the SWR1 and INO80 complexes catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosomes over the transcription start sites of genes6. However, many nucleosome-remodeling factors exhibit similar functions when assayed in vitro7. And, whereas some genes encoding nucleosome-remodeling factors are essential or required for robust viability in yeast, oth-ers can be deleted singly, without any major defect in viability, thus indicating functional redundancy8. Studies in yeast illustrate how various nucleosome-remodeling factors have overlapping and complementary functions to shape chromatin over and around genes, for example, in regulating the progression of RNA polymerase II9–12. Evidence for col-laboration between different remodelers is not restricted to gene regulation. Almost all classes of remodeler ATPases have been

The packaging of eukaryotic genomes into nucleosome-based chromatin necessitates mechanisms that regulate the access to DNA of, for example, the transcription machinery. The past ~20 years have revealed a plethora of different enzymes that modify chromatin, including chromatin- or nucleosome- remodeling enzymes that use energy gained from ATP hydrolysis to alter nucleosome struc-ture. Saccharomyces cerevisiae alone encodes eight factors with demonstrated nucleosome- remodeling activity similar to that of SWI2/SNF2 ATPase1. The picture is complicated further by the fact that a single SWI2/SNF2-like motor protein often forms several distinct complexes by stably interacting with other proteins2,3 (Fig. 1). These accessory subunits are involved in regulating the catalytic activity of the complexes; some of them bind histone-modifying enzymes or modified histones themselves, or are involved in recruitment by sequence-specific transcriptional regula-tors. In higher eukaryotes, there are many more nucleosome remodelers and distinct remodeling complexes formed around the same core ATPase2–4. Deletion of many of them leads to early embryonic lethality, and mutations in genes encoding these factors are implicated in several genetic diseases, most notably cancer2–5.

Chromatin remodeling: a collaborative effortPatrick D Varga-Weisz

Enzymes that alter nucleosome structure or position are at the very center of gene and genome regulation, and understanding how, and to what extent, these diverse activities collaborate and control each other to shape the genome for dynamic regulation is a major challenge. A new study provides an important step in this direction by illustrating the cooperative nature of ATP-dependent chromatin-remodeling systems in mammalian cells.

Patrick D. Varga-Weisz is at the Nuclear Dynamics Programme, Babraham Institute, Cambridge, UK. e-mail: [email protected]

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demonstrate co-occupancy (by sequential chromatin immunoprecipitation, ‘re-ChIP’), they concluded that they were detecting transient, sequential binding events rather than the formation of ‘super-remodeling complexes’ or simultaneous co-occupancy, for which evidence exists (for Chd4 and Snf2h26, and for CHD7 and PBAF23). Future experiments, for example using sharply inducible systems, should aim at elucidating in detail the interdependencies of the various remodeling factors.

It is interesting that another study per-formed in Drosophila cells came to dia-metrically different conclusions: distinct remodelers were found to have nonover-lapping genome-wide distributions, with little evidence for enrichment at promot-ers27. That study identified far fewer binding regions for each remodeler compared to those in the Morris et al.24 study. These distinct conclusions may reflect different biology

shared by all three remodelers and an even greater proportion shared by at least two. These findings suggest that remodelers may act in coordination at the same sites.

To examine the roles of remodelers in regu-lating chromatin accessibility, the authors gen-erated cell lines that express dominant-negative versions of either Brg1, Chd4 or Snf2h in a con-trollable manner. This approach showed that each remodeler renders some sites accessible while closing others. Many DHSs were affected by two or even all three remodelers, with evi-dence for synergistic and opposing actions by distinct remodelers. This study illustrates that multiple remodelers act over the same sites to shape chromatin and emphasizes the need to view chromatin dynamics as the action of multiple factors, possibly successively, over the same site (Fig. 2).

Because Morris et al.24 did not detect bio-chemical interactions between the distinct remodelers in cell extracts and could not

immuno precipitation and sequencing (ChIP-seq) to identify ~40,000 sites occu-pied by each remodeler, with the majority of the binding sites in the promoter regions and gene bodies. They focused on DNase I− hypersensitive sites (DHSs)—important higher-eukaryote chromatin features affected by nucleosome-remodeling factors—because these sites usually are markers of regulatory DNA elements, such as promoters, enhanc-ers, insulators, silencers and locus-control regions25. DHSs are generated through the activity of nucleosome remodelers—a notion that is strongly supported by Morris et al.24— and are areas where many transcriptional regulators congregate25. The authors found that a majority of remodeling factor−binding sites coincided with DHSs, in line with a link between remodelers and gene regulation. Most remarkably, they discovered that binding sites between remodelers showed a high degree of overlap, with more than 50% of sites being

Figure 1 Features of Brg1-, Chd4- and Snf2h-containing chromatin-remodeling complexes from mammalian cells. More comprehensive overviews are provided in several recent reviews1–4. (a) Key domains of the Brg1, Chd4 and Snf2h remodelers. (b) Noncatalytic subunits found in complexes formed around Brg1 (left), Chd4 (middle) and Snf2h (right). Slashes denote “and.” Additional details are in the aforementioned reviews1–4. (c) Chromatin-remodeling steps catalyzed by Brg1, Chd4 and Snf2h. Brg1-containing complexes can catalyze the disruption of nucleosomes (left). Complexes with all of these factors have been demonstrated to catalyze the sliding of nucleosomes (middle and right). Chd4-containing complexes comprise histone deacetylases HDAC1 and HDAC2 and mediate the ATP-dependent deacetylation of nucleosomes as well as nucleosome sliding (middle). Several Snf2h-containing complexes catalyze the regular ‘spacing’ of nucleosomes (right). Ac, acetyl group.

BAF60a–cBAF47

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Snf2h (ISWI family)

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ATPase ATPase ATPase

ACF1

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(for example, different cell types investigated or insect versus mammalian genomes), and this warrants further investigation.

Morris et al.24 provide an exciting glimpse into the complexity of chromatin dynamics and the role of multiple remod-elers in mammalian cells. It is a start of what will be a very challenging journey. Ultimately, the goal is to understand chro-matin-remodeling-factor dynamics and functions, genome wide, at high resolution in real time, possibly at the single-cell level. It will also be important to unravel the role of the various remodeling factors in determining the precise nucleosome positioning and histone- variant occupancy in mammalian cells, as has been done in yeast, because these features are crucial in gene regulation. Furthermore, it has been hypothesized that nucleosome alteration may not be the only aspect of chromatin- remodeling-factor function and that they may have noncatalytic roles, for example, in regulating chromatin-fiber interactions3. Thus, chromatin dynamics

need to be pictured in three and four dimen-sions. Although understanding all this seems to be an unattainable goal at pres-ent, the rapid advancement in technologies promises that at least some of these chal-lenges will be addressable in the not too distant future.

COMPETING FINANCIAL INTERESTSThe author declares no competing financial interests.

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Figure 2 Several scenarios illustrating how two distinct chromatin-remodeling complexes (complex 1 and complex 2) may regulate chromatin accessibility over the same site. (a) Two remodeling complexes simultaneously create an accessible site, for example, by forming a super-remodeling complex. (b) Remodeling complexes 1 and 2 act over the same site in a sequential, interdependent manner, either to create (left) or to close down a DHS (right), for example by shifting nucleosomes into the site. (c) One chromatin-remodeling factor opens a site that is then subsequently closed down by another one (for example by sliding nucleosomes) in a transient gene-activation process.

a b c Cooperative and synchronous Sequential and interdependent

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