The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h

Gamarra, Nathan; Johnson, Shirley; Trnka, Michael J.; Burlingame, Alma L.; Narlikar, Geeta J.

doi:10.7554/elife.35322

Cited by 69 publications

(87 citation statements)

References 58 publications

(134 reference statements)

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“…Experiments were performed as in (Gamarra et al, 2018), except that the imaging buffer was 53 mM HEPES-KOH, pH 7.5 at 22°C, 9.1 mM Tris-acetate, pH 7.5 at 22°C, 140 mM KCl, 0.5 mM MgCI 2 , 10% glycerol, 0.02% NP-40, 1% glucose, 0.1 mg/mL acetylated BSA, 2 mM Trolox, 0.03 mM β–mercaptoethanol, 2 U/μL catalase, and 0.08 U/μL glucose oxidase. SNF2h and ADP-BeF x were added simultaneously to a final concentration of 2 μM and 0.5 mM respectively using an automated syringe pump.…”

Section: Methodsmentioning

confidence: 99%

“…The linear phase of each reaction was then fit using linear regression using Prism to obtain hydrolysis rates. For nucleosome stimulated ATP hydrolysis, rates were measured using radioactivity as in (Gamarra et al, 2018) under saturating concentrations of nucleosomes without flanking DNA and subsaturating concentrations of (20μM) ATP-MgCl 2 . Reactions were performed in 12.5 mM HEPES pH 7.5, 70mM KCl, 3mM free MgCl 2 , 0.02% NP-40, and ~1.5% glycerol at 25°C, initiated by addition of enzyme, and time points quenched with an equal volume 50 mM Tris pH 7.5, 3% SDS, and 100 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…The other region of increased disorder is the acidic patch formed between histone H2A and H2B. Previous work has suggested that interactions between SNF2h and the acidic patch play a critical role in stimulating nucleosome sliding (Dann et al, 2017; Gamarra et al, 2018). Interestingly, recent biochemical studies using asymmetric acidic patch mutant nucleosomes indicate that the activity of a SNF2h protomer bound at SHL+2 (as defined in Figure 1C) requires the acidic patch on the undistorted octamer face (Face B) (G. Bowman & R.F.…”

Section: A Snf2h-nucleosome Complex With An Asymmetrically Deformed Hmentioning

confidence: 99%

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Electron cryo-microscopy structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome

Armache¹,

Gamarra²,

Sl³

et al. 2019

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18The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two 19 protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer 20 deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here 21 we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture 22 two reaction intermediates. In one structure, histone residues near the dyad and in the H2A- 23H2B acidic patch, distal to the active SNF2h protomer, are disordered. The disordered acidic 24 patch is expected to inhibit the second SNF2h promoter, while disorder near the dyad is 25 expected to promote DNA translocation. The other structure doesn't show octamer deformation, 26but surprisingly shows a 2bp translocation. FRET studies indicate that ADP-BeFx predisposes 27 SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for 28 allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric 29 octamer deformation to inhibit the second protomer, while stimulating directional DNA 30 translocation. 31 32 One sentence summary: Cryo-EM structures capture different conformational states of 33 chromatin remodeler-nucleosome complexes. 34 35 65 complex: a state with an unexpectedly translocated nucleosome (Figure 1, Figure 1-supplement 66 1-6), a state with two SNF2h protomers bound to a nucleosome (Figure 2A, Figure 2-67 supplement 1) and a state with one protomer bound to a nucleosome that shows increased 68 disorder within the histone core (Figure 2B). The locations of histone disorder strongly suggest a 69 4 role for octamer deformation in protomer coordination and directional DNA translocation. In 70 addition, we detect new ISWI-histone contacts that make significant contributions to 71 nucleosome sliding and help explain why ISWI may differ in mechanism from Swi2/Snf2 (Figure 72 3) (Liu et al., 2017).73 74 Overview of SNF2h-nucleosome structures 75 Like most ISWI remodelers, SNF2h slides mono-nucleosomes assembled on short stretches of 76 DNA towards the center of the DNA (Clapier and Cairns, 2009; Narlikar et al., 2013; Zhou et al., 77 2016). In previous studies we have found that while a monomer of SNF2h can slide 78 nucleosomes, SNF2h functions most optimally as a dimer (Leonard and Narlikar, 2015; Racki et 79 al., 2009). In these studies, we were able to visualize both singly bound and doubly bound 80 SNF2h using negative stain EM (Racki et al., 2009). Previous studies have further shown that 81 binding of the ATP analog, ADP-BeF x , promotes a restricted conformation of the ATPase active 82 site in a manner that is dependent on the H4 tail (Racki et al., 2014). The restricted 83 conformation is consistent with observations showing an activating role for the H4 tail (Clapier et 84 al., 2001; 2002; Hamiche et al., 2001). Further, binding of ADP-BeF x to SNF2h promotes 85 conformational flexibility of buried histone residues (Sinha et al., 2017). This conformational 86 flexibility is functionally important ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: A Snf2h-nucleosome Complex With An Asymmetrically Deformed Hmentioning

confidence: 99%

See 1 more Smart Citation

Electron cryo-microscopy structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome

Armache¹,

Gamarra²,

Sl³

et al. 2019

Preprint

Self Cite

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“…Cross-linked peptides were desalted, fractionated by size-exclusion chromatography (SEC), and analyzed by LC-MS using a previously described method 66 . Briefly, trypsin digests were acidified to 0.2% TFA, desalted, and run over a Superdex Peptide PC 3.2/300 SEC column (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

Sanulli

Trnka

Dharmarajan

et al. 2018

Preprint

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20The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet 21 formation. Yet, a connection between phase separation and chromatin compaction has not been 22 experimentally demonstrated. More fundamentally, how HP1 action at the level of a single 23 nucleosome drives chromatin compaction remains poorly understood. Here we directly 24 demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes 25 into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-26 spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of 27 buried histone residues within a mononucleosome. Restraining these dynamics via site-specific 28 disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. 29Our results indicate that chromatin compaction and phase separation can be highly coupled 30 processes. Further, we find that such coupling is promoted by a counter-intuitive function of 31 Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by 32 weak and dynamic multivalent interactions. We propose that dynamic exposure of buried 33 histone residues increases opportunities for multivalent interactions between nucleosomes, 34 thereby coupling chromatin compaction to phase separation. We anticipate that this new model 35 for chromatin organization may more generally explain the formation of highly compacted 36 chromatin assemblies beyond heterochromatin. 37 38 39 Main text: 40 Heterochromatin formation impacts genome function at multiple scales. Thus heterochromatin 41 enables heritable gene repression, helps maintain chromosome integrity and provides 42 mechanical rigidity to the nucleus 1,2 . It has been proposed that these diverse functions arise in 43 part from compaction of the underlying chromatin. Yet how heterochromatin proteins enable 44 chromatin compaction remains poorly understood. 46A major type of heterochromatin contains at its core the complex formed between HP1 proteins 47 and chromatin that is methylated on histone H3, lysine 9 (H3K9me) 3 . Previous work has 48 suggested that dimerization and higher-order oligomerization of HP1 proteins helps promote 49 chromatin compaction 4,5 . More recently it has been shown that human and Drosophila HP1 50 proteins can form phase-separated droplets and that chromatin can get incorporated into 51 droplets formed by the human HP1 protein HP1α 6,7 . These results have led to the model that 52 phase separation mediated by HP1 proteins compartmentalizes and compacts chromatin.Further, it has long been theorized that macromolecular crowding within the nucleus may lead to 54 phase separation of chromatin 8 . However, two types of fundamental questions have remained 55 unanswered: (1) It is poorly understood how HP1 proteins regulate chromatin structure to 56 enable its compaction and, (2) it is not clear whether and how chromatin compaction 57 mechanisms are related to phase separation. Importantly, exper...

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“…These proteins include the Latent Nuclear Antigen peptide (LANA) of Kaposi's sarcoma virus, the H2A ubiquitylation module of Polycomb Repressive Complex 1, and the H2B deubiquitylation module of the SAGA coactivator complex (2)(3)(4)(5)(6). Through direct interactions, the acidic patch also regulates the binding and activities of ATP-dependent chromatin remodeling enzymes (7,8). Mutations that disrupt the acidic patch impair the binding and functions of proteins important for transcription (4,9), DNA damage repair (10,11), and chromosome segregation (12), and a recent search for mutations in gynecological carcinomas identified a missense mutation in the human gene HIST1H2AB, which leads to a substitution, H2A-E57Q, in a conserved acidic patch residue (13).…”

Section: Introductionmentioning

confidence: 99%

The nucleosome acidic patch directly interacts with subunits of the Paf1 and FACT complexes and controls chromatin architecture in vivo

Cucinotta

Hildreth²,

McShane³

et al. 2019

Preprint

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The nucleosome core regulates DNA-templated processes through the highly conserved nucleosome acidic patch. While structural and biochemical studies have shown that the acidic patch controls chromatin factor binding and activity, few studies have elucidated its functions in vivo. We employed site-specific crosslinking to identify proteins that directly bind the acidic patch in Saccharomyces cerevisiae and demonstrated crosslinking of histone H2A to Paf1 complex subunit Rtf1 and FACT subunit Spt16. Rtf1 bound to nucleosomes through its histone modification domain, supporting its role as a cofactor in H2B K123 ubiquitylation. An acidic patch mutant showed defects in nucleosome positioning and occupancy genome-wide. Our results provide new information on the chromatin engagement of two central players in transcription elongation and emphasize the importance of the nucleosome core as a hub for proteins that regulate chromatin during transcription.Cucinotta et al.

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The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h

Cited by 69 publications

References 58 publications

Electron cryo-microscopy structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome

Electron cryo-microscopy structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

The nucleosome acidic patch directly interacts with subunits of the Paf1 and FACT complexes and controls chromatin architecture in vivo

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